Mao Xia

Mitophagy Enhances Oncolytic Measles Virus Replication by Mitigating DDX58/RIG-I-Like Receptor Signaling

ABSTRACT The success of future clinical trials with oncolytic viruses depends on the identification and the control of mechanisms that modulate their therapeutic efficacy. In particular, little is known about the role of autophagy in infection by attenuated measles virus of the Edmonston strain (MV-Edm). We investigated the interaction between autophagy, innate immune response, and oncolytic activity of MV-Edm, since the antiviral immune response is a known factor limiting virotherapies. We report that MV-Edm exploits selective autophagy to mitigate the innate immune response mediated by DDX58/RIG-I like receptors (RLRs) in non-small cell lung cancer (NSCLC) cells. Both RNA interference (RNAi) and overexpression approaches demonstrate that autophagy enhances viral replication and inhibits the production of type I interferons regulated by RLRs. We show that MV-Edm unexpectedly triggers SQSTM1/p62-mediated mitophagy, resulting in decreased mitochondrion-tethered mitochondrial antiviral signaling protein (MAVS) and subsequently weakening the innate immune response. These results unveil a novel infectious strategy based on the usurpation of mitophagy leading to mitigation of the innate immune response. This finding provides a rationale to modulate autophagy in oncolytic virotherapy. IMPORTANCE In vitro studies, preclinical experiments in vivo, and clinical trials with humans all indicate that oncolytic viruses hold promise for cancer therapy. Measles virus of the Edmonston strain (MV-Edm), which is an attenuated virus derived from the common wild-type measles virus, is paradigmatic for therapeutic oncolytic viruses. MV-Edm replicates preferentially in and kills cancer cells. The efficiency of MV-Edm is limited by the immune response of the host against viruses. In our study, we revealed that MV-Edm usurps a homeostatic mechanism of intracellular degradation of mitochondria, coined mitophagy, to attenuate the innate immune response in cancer cells. This strategy might provide a replicative advantage for the virus against the development of antiviral immune responses by the host. These findings are important since they may not only indicate that inducers of autophagy could enhance the efficacy of oncolytic therapies but also provide clues for antiviral therapy by targeting SQSTM1/p62-mediated mitophagy.

Multifunctional antitumor molecule 5′-triphosphate siRNA combining glutaminase silencing and RIG-I activation

Resisting cell death, reprogrammed metabolism and immune escape are fundamental traits of hard‐to‐treat cancers. Therapeutic improvement can be expected by designing drugs targeting all three aspects. 5′‐Triphosphate RNA (ppp‐RNA), a specific ligand of the pattern recognition receptor retinoic acid‐inducible gene I (RIG‐I), has been shown to trigger intrinsic apoptosis of malignant cells and to activate antitumor immune responses via type I interferons (IFNs). In our study, we designed a ppp‐modified siRNA specifically silencing glutaminase (ppp‐GLS), a key enzyme of glutaminolysis that is indispensable for many cancer types. Bifunctional ppp‐GLS induced more prominent antitumor responses than RNA molecules that contained either the RIG‐I ligand motif or GLS silencing capability alone. The cytopathic effect was constrained to tumor cells as nonmalignant cells were not affected. We then analyzed the mechanisms leading to the profound antitumor efficacy. First, ppp‐GLS effectively induced intrinsic proapoptotic signaling. In addition, GLS silencing sensitized malignant cells to RIG‐I‐induced apoptosis. Moreover, disturbed glutaminolysis by GLS silencing contributed to enhanced cytotoxicity. Finally, RIG‐I activation blocked autophagic degradation leading to dysfunctional mitochondria and reactive oxygen species (ROS) generation, whereas GLS silencing severely impaired ROS scavenging systems, leading to a vicious circle of ROS‐mediated cytotoxicity. Taken together, ppp‐GLS combines cell death induction, immune activation and glutaminase inhibition in a single molecule and has high therapeutic efficacy against cancer cells.

One-Step Photo Synthesis of Protein–Drug Nanoassemblies for Drug Delivery

Upon controlled UV illumination, disulfide bonds in bovine α-lactalbumin (BLA) are selectively broken, leading to self-assembly of the BLA and doxorubicin (DOX) molecules into nanoparticles via hydrophobic interactions and intermolecular disulfide bonds. Such protein-drug nanoparticles have synergistic anticancer activity in vitro and tumor-homing specificity in vivo, which are of great potential for systemic drug delivery in cancer therapy.

Photo synthesis of protein-based drug-delivery nanoparticles for active tumor targeting

Recently, nanometer-sized drug-delivery systems (DDSs) with an active targeting function to tumor sites have been cinfirmed to have great efficiency for cancer chemotherapy. However, the use of complicated modification techniques and the poor graft efficiency of the targeting ligands on the surface of the DDS have largely limited the application of most of the nano-DDSs. Especially for protein-based nano-DDSs, grafting of targeting ligands often requires genetic modification of the protein sequences, which is laborious and inefficient. Here, we present a novel method to photo synthesize a protein-based drug-delivery system with an active targeting function to tumor sites. The disulfide bonds in protein bovine α-lactalbumin (BLA) can be ruptured by controlled UV illumination, which triggers the formation of nano-sized protein aggregates and releases free thiol groups for the modification of the active targeting ligand of circular RGD peptide. Moreover, we demonstrated that the anti-cancer drug doxorubicin (DOX) can be loaded onto the protein-based DDS during the photo synthesis step. The synthesis approach is very convenient and cost-effective and can be accomplished under physiological conditions. Both in vitro and in vivo experiments validate that this DDS system possesses a much greater drug-delivery efficiency to the tumor sites and a better inhibition capability of tumor growth than the unmodified counterparts. This novel drug-delivery system can find broad applications in cancer chemotherapy. Moreover, our synthetic strategy can be easily extended to other disulfide-containing proteins and greatly expand the tool-box of protein-based DDSs for active targeting.

Photoacoustic “nanobombs” fight against undesirable vesicular compartmentalization of anticancer drugs

Undesirable intracellular vesicular compartmentalization of anticancer drugs in cancer cells is a common cause of chemoresistance. Strategies aimed at circumventing this problem may improve chemotherapeutic efficacy. We report a novel photophysical strategy for controlled-disruption of vesicular sequestration of the anticancer drug doxorubicin (DOX). Single-walled carbon nanotubes (SWCNTs), modified with folate, were trapped in acidic vesicles after entering lung cancer cells. Upon irradiation by near-infrared pulsed laser, these vesicles were massively broken by the resulting photoacoustic shockwave, and the vesicle-sequestered contents were released, leading to redistribution of DOX from cytoplasm to the target-containing nucleus. Redistribution resulted in 12-fold decrease of the EC50 of DOX in lung cancer cells, and enhanced antitumor efficacy of low-dose DOX in tumor-bearing mice. Side effects were not observed. These findings provide insights of using nanotechnology to improve cancer chemotherapy, i.e. not only for drug delivery, but also for overcoming intracellular drug-transport hurdles.

Oncolytic measles virus enhances antitumour responses of adoptive CD8 + NKG2D + cells in hepatocellular carcinoma treatment

There is an urgent need for novel effective treatment for hepatocellular carcinoma (HCC). Oncolytic viruses (OVs) not only directly lyse malignant cells, but also induce potent antitumour immune responses. The potency and precise mechanisms of antitumour immune activation by attenuated measles virus remain unclear. In this study, we investigated the potency of the measles virus vaccine strain Edmonston (MV-Edm) in improving adoptive CD8+NKG2D+ cells for HCC treatment. We show that MV-Edm-infected HCC enhanced the antitumour activity of CD8+NKG2D+ cells, mediated by at least three distinct mechanisms. First, MV-Edm infection compelled HCC cells to express the specific NKG2D ligands MICA/B, which may contribute to the activation of CD8+NKG2D+ cells. Second, MV-Edm-infected HCC cells stimulated CD8+NKG2D+ cells to express high level of FasL resulting in enhanced induction of apoptosis. Third, intratumoural administration of MV-Edm enhanced infiltration of intravenously injected CD8+NKG2D+ cells. Moreover, we found that MV-Edm and adoptive CD8+NKG2D+ cells, either administered alone or combined, upregulated the immune suppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in HCC. Elimination of IDO1 by fludarabine enhanced antitumour responses. Taken together, our data provide a novel and clinically relevant strategy for treatment of HCC.

Anti-angiogenic efficacy of 5'-triphosphate siRNA combining VEGF silencing and RIG-I activation in NSCLCs.

Short interfering RNA (siRNA) targeting angiogenic factors and further inhibiting tumor angiogenesis, is one of the potent antitumor candidates for lung cancer treatment. However, this strategy must be combined with other therapeutics like chemotherapy. In this study, we designed a 5′-triphosphate siRNA targeting VEGF (ppp-VEGF), and showed that ppp-VEGF exerted three distinct antitumor effects: i) inhibition of tumor angiogenesis by silencing VEGF, ii) induction of innate immune responses by activating RIG-I signaling pathway, and thus activate antitumor immunity, iii) induction of apoptosis. In a subcutaneous model of murine lung cancer, ppp-VEGF displayed a potent antitumor effect. Our results provide a multifunctional antitumor molecule that may overcome the shortages of traditional antiangiogenic agents.

Carrier Cells for Delivery of Oncolytic Measles Virus into Tumors: Determinants of Efficient Loading

AbstractOncolytic measles virus (OMV) is a promising antitumor agent. However, the presence of anti-measles neutralizing antibodies (NAbs) against the hemagglutinin (H) protein of OMV is a major barrier to the therapeutic application of OMV in clinical practice. In order to overcome this challenge, specific types of cells have been used as carriers for OMV. Differential loading strategies appear to result in different therapeutic outcomes; despite this, only few studies have reported practical ex vivo loading strategies required for effective treatment. To this end, we systematically evaluated the antitumor efficacy of OMV using different loading strategies; this involved varying the in vitro loading duration and loading dose of OMV. We found that improved oncolysis of carrier cells was achieved by a prolonged loading duration in the absence of NAbs. However, the enhanced oncolytic effect was abrogated in the presence of NAbs. Further, we found that the expression of H protein on the surface of carrier cells was predominantly determined by the loading duration rather than the loading dose. Finally, we showed that NAbs blocked viral transfer by targeting H protein prior to the occurrence of cell-to-cell interactions. Our results provide comprehensive information on the determinants of an effective loading strategy for carrier cell-based virotherapy; these results may be useful for guiding the application of OMV as an antitumor agent in clinical practice.