Protein kinase inhibition experiments established that signaling pathways involved in control over translation modulate PVSRIPO translation, replication and cell killing. Blocking the activity of the downstream eIF4E kinase and Erk1/2 substrate, MAPK signal-integrating kinase 1 (Mnk1) – using the Mnk1 inhibitor CGP57380 – depresses PVSRIPO translation, proliferation and cell killing in GBM cells ()
]. Interestingly, in PVSRIPO nonpermissive HEK293 cells, Ras–Erk signaling is inherently low ()
]. In fact, the relative levels of p-Erk1/2 and p-eIF4E in HEK293 versus GBM cells resembles those in normal human brain versus patient GBM tissues ()
]. Activating Ras–Erk signaling in HEK293 cells by introducing a tetracycline-inducible form of oncogenic Ras induced phosphorylation of Erk1/2 and eIF4E to levels comparable with GBM cells or patients’ tumors ()
. This was accompanied by significantly enhanced PVSRIPO replication ()
]. These studies implicated Ras–Erk signals in PVSRIPO translation, propagation and tumor cell killing. We fine-tuned our approach to investigate the precise molecular events responsible for mediating PVSRIPO cell killing. Introducing constitutively active Mnk1 into HEK293 cells, which are naturally resistant to PVSRIPO cytotoxicity [49
], mediates enhanced viral cytotoxicity. Conversely, a dominant-negative Mnk1 variant had the opposite effect [61
]. Mnk1 binding to eIF4G [63
], which is strongly responsive to Ras–Erk activation [64
], results in phosphorylation of Ser209 in eIF4E [65
]. It therefore appears that downstream Ras–Erk signals converging on Mnk1, its binding partner eIF4G and its substrate eIF4E control susceptibility to PVSRIPO in cancer cells. This may involve differential regulation of eIF4G’s ability to bind to the IRES in PVSRIPO (see following section).
Protein kinase inhibitors modulate PVSRIPO oncolysis in glioblastoma cells
Oncogenic H-Ras rescues PVSRIPO growth in nonpermissive HEK293 cells
Inhibition of the PI3K pathway (using the PI3K inhibitor LY294002) slightly, but reproducibly, enhanced PVSRIPO translation, replication and cell killing in GBM ()
]. In addition, treating GBM xenografts with PVSRIPO in combination with PI3K inhibitors led to accelerated loss of viable tumor [61
]. While mTORC1 inhibition modestly enhances PVSRIPO translation, replication and tumor cell killing in vitro
and in vivo
, Ras–Erk signaling pathways have a decisive influence over PVSRIPO oncolysis ()
]. This may indicate that Erk1/2 signaling to the protein synthesis apparatus exerts dominant effects on the regulation of cap-independent translation in cancer cells.
Mechanistic basis for PVSRIPO dependence on mitogenic signal transduction
Several proposed oncolytic viruses share a bias towards certain signaling pathways. For example, inhibition of mTORC1 stimulates vesicular stomatitis virus-mediated tumor cell killing by impeding mTORC1-dependent cytokine responses [66
]. Since mitogenic signaling pathways are pleiotropic and host cell cytotoxicity of oncolytic viruses is influenced by multiple factors, unraveling the mechanistic basis for the effect of signal transduction on viral cancer cell killing can be daunting. In this regard, poliovirus’ singular dependence on a specific translation initiation event (binding of eIF4G to the IRES) at a defined moment during the viral life-cycle (early after uncoating of the viral RNA) presents a uniquely simple scenario. As outlined previously, the virus’ strategy to combat host cell defenses and unleash viral translation and propagation depend on immediate cap-independent translation of viral nonstructural proteins. Thus, the events controlling ribosome recruitment (via eIF4G) at the incoming viral genome may determine the outcome of the infection. It is obvious that once 2Apro is expressed, host eIF4G is cleaved and the host cell has been subverted, the activity of specific signal transduction pathways no longer matter to the outcome of infection.
The factors determining eIF4G binding to viral IRESs are not immediately clear. Although eIF4G is a confirmed RNA-binding protein [67
], it lacks a classic RNA recognition motif or other defined structures known to mediate RNA binding. Similarly, IRESs are defined by their function, not their structure. IRESs known to initiate translation via direct recruitment of eIF4G (e.g., the IRESs of poliovirus [46
], the c-myc
oncogene mRNA [68
] or the VEGF
]) have no apparent structural similarities. Therefore, there is no defined RNA structure or motif that can be examined to determine a capacity for eIF4G binding.
According to phosphoproteomic screens, eIF4G has approximately 17 mitogen-responsive phosphorylation sites [70
]. It is thus conceivable that signal transduction to eIF4G (e.g., via Ras–Erk) leads to phosphorylation events that alter its RNA-binding properties. Despite strong evidence for phosphorylation of eIF4G upon activation of mitogenic signal transduction pathways [71
] and a prominent role for eIF4G in protein synthesis regulation, no specific kinases or signal transduction pathways converging on eIF4G have been identified. In addition, the biological effects of signaling to eIF4G remain a mystery. Signal transduction to eIF4G and its effect on translation regulation (e.g., via the poliovirus IRES) is an active area of investigation in our laboratory. While it is too early to communicate definitive insight into this difficult regulatory system, we propose a number of hypotheses.
First, a cluster of mitogen-response phosphorylation sites in eIF4G maps to the ‘interdomain linker’, a flexible loop connecting HEAT domains 1 and 2 of eIF4G [72
]. This region also contains the RNA-binding determinants of eIF4G [67
]. It is thus conceivable that phosphorylation of residues in the eIF4G interdomain linker influence cap-independent translation by regulating eIF4G’s ability to bind to IRESs in target mRNAs.
Second, our research suggests that Ras–Erk activation controls the association of eIF4G with Mnk1 [73
]. Binding of Mnk1 to eIF4G is essential for phosphorylation of the Mnk1 substrate, eIF4E [63
]. It is currently not understood whether Mnk1 binding to eIF4G alone modulates eIF4G function (e.g., by altering the RNA-binding capacity).
Third, the availability of eIF4G for IRES-mediated translation may be codetermined by the eIF4E binding proteins (BPs) ()
]. Activity of the eIF4E-BPs is controlled by mTORC1. In the nonphosphorylated state, the eIF4E-BPs bind to eIF4E and prevent its association with eIF4G, leading to repression of cap-dependent translation [75
]. Activation of mTORC1, the eIF4E-BP kinase, leads to eIF4E-BP hyperphosphorylation, dissociation from eIF4E and stimulation of cap-dependent translation [76
]. It is well established that inhibitors of the PI3K–mTORC1 signaling pathway, in addition to repression of cap-dependent translation, induce alternative, cap-independent protein synthesis. For example, the classic mTORC1 inhibitor rapamycin stimulates WT poliovirus replication [77
]. The mechanism responsible for this effect may be that rapamycin-induced eIF4E-BP dephosphorylation enhances the proportion of ‘free’ (not committed in m7G-cap binding complexes) eIF4G. This may result in enhanced IRES binding of eIF4G [74