Activated ras signaling pathways and reovirus oncolysis: an update on the mechanism of preferential reovirus replication in cancer cells

Abstract

The development of wild-type, unmodified Type 3 Dearing strain reovirus as an anticancer agent has currently expanded to 32 clinical trials (both completed and ongoing) involving reovirus in the treatment of cancer. It has been more than 30 years since the potential of reovirus as an anticancer agent was first identified in studies that demonstrated the preferential replication of reovirus in transformed cell lines but not in normal cells. Later investigations have revealed the involvement of activated Ras signaling pathways (both upstream and downstream) and key steps of the reovirus infectious cycle in promoting preferential replication in cancer cells with reovirus-induced cancer cell death occurring through necrotic, apoptotic, and autophagic pathways. There is increasing evidence that reovirus-induced antitumor immunity involving both innate and adaptive responses also contributes to therapeutic efficacy though this discussion is beyond the scope of this article. Here, we review our current understanding of the mechanism of oncolysis contributing to the broad anticancer activity of reovirus. Further understanding of reovirus oncolysis is critical in enhancing the clinical development and efficacy of reovirus.

Keywords: EGFR; PKR; Ras; apoptosis; autophagy; necrosis; oncolysis; reovirus.

Figure 1

Reovirus structure and infectious cycle. (A) Reovirus is a non-enveloped double-stranded RNA (dsRNA) virus approximately 80 nm in diameter. The viral genome, consisting of 10 segments of dsRNA, is contained within an outer and inner capsid and encodes for structural proteins comprising the outer capsid including sigma 1 (σ1), sigma 3 (σ3), lambda 2 (λ2), and mu 1 (μ1), structural proteins comprising the inner capsid including sigma 2 (σ2), lambda 1 (λ1), lambda 3 (λ3), and mu 2 (μ2), and non-structural proteins including sigma 1s (σ1s), sigma NS (σNS), mu NS (μNS), and mu NSC (μNSC). σ1 has been identified as the viral attachment protein, μ1, μ2, and λ3 serve roles in viral replication, σ1s and σ3 appear to have roles in virulence, and σNS, μNS, and μNSC appear to be involved in the formation of viral inclusions. Figure reproduced with permission from ViralZone, SIB Swiss Institute of Bioinformatics. (B) Viral attachment to host cell surface glycans results in internalization of reovirus via receptor-mediated endocytosis. Alternatively, infectious subvirion particles (ISVPs) can be formed from proteolysis by extracellular proteases allowing their direct entry into cells via membrane penetration. Once internalized, the virus is transported to early and late endosomes where it undergoes proteolytic disassembly and degradation resulting in the formation of ISVPs and subsequently in the release of transcriptionally active viral core particles into the cytoplasm. Activated RNA-dependent RNA polymerase begins primary transcription within the core particles resulting in the release of primary transcripts that, along with protein products of early translation, form complexes or inclusions where further transcription and translation occur which, in turn, ultimately lead to viral replication and assembly, host cell death, and progeny release.

Figure 2

Ras-transformation affects several steps of the reovirus infectious cycle to promote oncolysis. Ras-transformation affects multiple steps of the infectious life cycle in promoting reovirus oncolysis by: (1) enhancing virus uncoating and disassembly, (2) negative regulation of retinoic acid-inducible gene I (RIG-I) signaling and releasing dsRNA-activated protein kinase (PKR)-induced translational inhibition, (3) increasing progeny release through enhanced apoptosis and generating more infectious progeny, and (4) enhancing viral spread in subsequent rounds of infection. Reovirus-induced cancer cell death occurs through autophagic, apoptotic, and necrotic pathways. Programed necrosis or necroptosis occurs through binding of tumor necrosis factor-α (TNF-α), Fas ligand (Fas), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to death receptors leading to downstream signaling involving receptor interaction protein kinase (RIP) 1 and 3, cylindromatosis (CYLD), TNF receptor-associated factors (TRAFs), stress-activated c-Jun NH₂-terminal protein kinase (JNK), reactive oxygen species (ROS), adenine nucleotide translocase (ANT), poly ADP-ribose polymerases (PARPs), phospholipases, and lipoxygenases (LOXs). Apoptosis occurs through both extrinsic [e.g., TRAIL binding to cell surface death receptor recruits Fas-associated death domain (FADD), which recruits and activates the initiator caspase-8 that ultimately activates effector caspases-3 and -7] and intrinsic pathways [cytochrome c and second mitochondrion-derived activator of caspase (Smac/DIABLO) release with activation of downstream effector caspases with or without caspase-9]. Autophagy occurs through endoplasmic reticulum (ER) stress and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)-mediated signaling. Dashed arrows represent putative cross-talk and suggested signaling pathways. EGFR, epidermal growth factor receptor; SOS, son of sevenless; RalGEF, guanine nucleotide exchange factors (GEFs) for the small G protein Ral pathways; MEK, mitogen-activated protein kinase (MAPK) kinase; ERK, extracellular signal regulated kinase; TLRs: toll-like receptors; MDA5, melanoma differentiation-associated protein 5; IFN-β, interferon-beta; eIF-2α, eukaryotic initiation factor 2α; NF-κB, nuclear factor kappa light-chain enhancer of activated B cells; IRF-3, interferon regulatory factor 3.