Yichen Wang

Targeting CD47 and Autophagy Elicited Enhanced Antitumor Effects in Non–Small Cell Lung Cancer

Blocking CD47 interactions was a potent antitumor therapy for NSCLC. Cells resist death by increasing autophagy; simultaneously inhibiting autophagy provided a synergistic antitumor effect, providing a scientific basis for enhancing the efficacy of immune checkpoint inhibitors. CD47-specific antibodies and fusion proteins that block CD47–SIRPα signaling are employed as antitumor agents for several cancers. Here, we investigated the synergistic antitumor effect of simultaneously targeting CD47 and autophagy in non–small cell lung cancer (NSCLC). SIRPαD1-Fc, a novel CD47-targeting fusion protein, was generated and was found to increase the phagocytic and cytotoxic activities of macrophages against NSCLC cells. During this process, autophagy was markedly triggered, which was characterized by the three main stages of autophagic flux, including formation and accumulation of autophagosomes, fusion of autophagosomes with lysosomes, and degradation of autophagosomes in lysosomes. Meanwhile, reactive oxygen species and inactivation of mTOR were shown to be involved in autophagy initiation in SIRPαD1-Fc–treated cells, indicating a probable mechanism for autophagy activation after targeting CD47 by SIRPαD1-Fc. Inhibition of autophagy enhanced macrophage-mediated phagocytosis and cytotoxicity against SIRPαD1-Fc–treated NSCLC cells. In addition, simultaneously targeting both CD47 and autophagy in NSCLC xenograft models elicited enhanced antitumor effects, with recruitment of macrophages, activated caspase-3, and overproduction of ROS at the tumor site. Our data elucidated the cytoprotective role of autophagy in CD47-targeted therapy and highlighted the potential approach for NSCLC treatment by simultaneously targeting CD47 and autophagy. Cancer Immunol Res; 5(5); 363–75. ©2017 AACR. See related Spotlight by Kaufman, p. 355.

Disrupting CD47-SIRPα axis alone or combined with autophagy depletion for the therapy of glioblastoma

CD47-targeting immune checkpoint inhibitors have been investigated for immunotherapy of several cancers, glioblastoma, one of the most common tumors in brain, was still a challenge for CD47-targeting therapy. Herein, we reported novel strategies for glioblastoma therapy via blocking CD47-signal regulatory protein-α (SIRPα) by SIRPα-Fc alone or in combination with autophagy inhibition. Our results showed that SIRPα-Fc increased macrophages-triggered cytotoxicity and phagocytosis of glioblastoma cells then elicited potent anti-tumor efficacy. During the treatment, SIRPα-Fc induced autophagy and autophagic flux in glioblastoma cells and Akt/mammalian target of rapamycin (mTOR) inactivation was participated in the autophagy activation. Inhibition of autophagy by pharmacological agents or small-interfering RNA increased SIRPα-Fc-triggered macrophage phagocytosis and cytotoxicity. Importantly, when compared with SIRPα-Fc treatment, blocking both CD47/SIRPα and autophagy significantly increased infiltration of macrophages and apoptosis of tumor cells, triggering potentiated anti-glioblastoma effect and extended median survival. Further experiments showed that adaptive immune response, including CD8+ T-cell subsets, was also played a crucial role in SIRPα-Fc-induced glioblastoma rejection. Our results indicated that SIRPα-Fc alone or combined with autophagy inhibitors elicited potent anti-glioblastoma effect, highlighting potential therapeutic strategies of glioblastoma via blocking CD47/SIRPα alone or in combination with autophagy inhibitor.

Tethering Interleukin-22 to Apolipoprotein A-I Ameliorates Mice from Acetaminophen-induced Liver Injury

Increasing evidence indicates that interleukin-22 (IL-22) holds tremendous potential as a protective agent in preventing liver injury, but its pleiotropic effects and pathogenic role in carcinogenesis, rheumatoid arthritis and psoriasis restrict its systemic application. Here, we first developed a nanoparticle (liposIA) as a liver-targeted agent through IL-22 tethered to apolipoprotein A-I (ApoA-I) in a gene therapy vector. LiposIA was prepared using thin film dispersion method and the complexes exhibited desirable nanoparticle size, fine polydisperse index, highly efficient transfection, and excellent serum and storage stability. Biodistribution and hepatic STAT3 phosphorylation studies revealed that IL-22 tethered to ApoA-I led to highly efficient liver targeting. More importantly, our studies showed that a single-dose of liposIA was able to protect mice against acetaminophen-induced liver injury and did not initiate inflammatory response or systemic toxicity in vivo. During this process, activated STAT3/Erk and Akt/mTOR signaling transductions were observed, as well as inhibition of reactive oxygen species (ROS) generation, which prevented mitochondrial dysfunction. These studies demonstrated that IL-22 tethered to apolipoprotein A-I could target and ameliorate acetaminophen-induced acute liver injury, which highlighted that a targeted strategy for IL-22 delivery might have broad utility for the protection of hepatocellular damage.

Interleukin-22 ameliorated renal injury and fibrosis in diabetic nephropathy through inhibition of NLRP3 inflammasome activation

Diabetic nephropathy (DN) is one of the most lethal complications of diabetes mellitus with metabolic disorders and chronic inflammation. Although the cytokine IL-22 was initially implicated in the pathogenesis of chronic inflammatory diseases, recent studies suggested that IL-22 could suppress inflammatory responses and alleviate tissue injury. Herein, we examined the role of IL-22 in DN. We found that serum levels of IL-22 were significantly downregulated in both patients and mice with DN. The expression of IL-22 was further decreased with the progression of DN, whereas IL-22 gene therapy significantly ameliorated renal injury and mesangial matrix expansion in mice with established nephropathy. IL-22 could also markedly reduce high glucose-induced and TGF-β1-induced overexpression of fibronectin and collagen IV in mouse renal glomerular mesangial cells in a dose-dependent manner, suggesting the potential role of IL-22 to inhibit the overproduction of ECM in vitro. Simultaneously, IL-22 gene therapy drastically alleviated renal fibrosis and proteinuria excretion in DN. In addition, IL-22 gene therapy markedly attenuated hyperglycemia and metabolic disorders in streptozotocin-induced experimental diabetic mice. Notably, IL-22 drastically reversed renal activation of NLRP3, cleavage of caspase-1, and the maturation of IL-1β in DN, suggesting unexpected anti-inflammatory function of IL-22 via suppressing the activation of NLRP3 inflammasome in vivo. Moreover, IL-22 markedly downregulated high glucose-induced activation of NLRP3 inflammasome in renal mesangial cells in a dose-dependent manner, indicating that the effects of IL-22 on NLRP3 inflammasome activation was independent of improved glycemic control. These results suggested that nephroprotection by IL-22 in DN was most likely associated with reduced activation of NLRP3 inflammasome. In conclusion, our finding demonstrated that IL-22 could exert favorable effects on DN via simultaneously alleviating systemic metabolic syndrome and downregulating renal NLRP3/caspase-1/IL-1β pathway, suggesting that IL-22 might have therapeutic potential for the treatment of DN.

NOD-Like Receptor Protein 3 Inflammasome-Dependent IL-1β Accelerated ConA-Induced Hepatitis

Autoimmune hepatitis (AIH) is a progressive inflammatory disorders of unknown etiology, characterized by immune-mediated destruction of hepatocytes and massive production of cytokines. Interleukin-1β is a pleiotropic proinflammatory cytokine and well known to be critical in a variety of autoimmune diseases. However, the role of interleukin-1β (IL-1β) in AIH is still indistinct. Here, we first investigated the significance of NOD-like receptor protein 3 (NLRP3) inflammasome-dependent IL-1β in the pathogenesis of AIH with a murine model of immune-mediated hepatitis induced by Concanavalin A (ConA). In ConA-treated mice, pathogenic elevated NLRP3, Cleaved caspase-1 and IL-1β levels, as well as an inflammatory cell death known as pyroptosis predominantly occurred in the livers. Strikingly, NLRP3−/− and caspase-1−/− mice were broadly protected from hepatitis as determined by decreased histological liver injury, serum aminotransferase (ALT)/aspartate transaminase levels, and pyroptosis. In vivo intervention with recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) strongly suppressed ConA-induced hepatitis by decreasing tumor necrosis factor-alpha (TNF-α) and interleukin-17 (IL-17) secretion, and inflammatory cell infiltration into livers. Additionally, rhIL-1Ra-pretreated mice developed significantly reduced NLRP3 inflammasome activation and reactive oxygen species (ROS) generation. Scavenging of ROS by N-acetyl-cysteine also attenuated NLRP3 inflammasome activation and liver inflammation, indicating that the essential role of ROS in mediating NLRP3 inflammasome activation in ConA-induced hepatitis. In conclusion, our results demonstrated that NLRP3 inflammasome-dependent IL-1β production was crucial in the pathogenesis of ConA-induced hepatitis, which shed light on the development of promising therapeutic strategies for AIH by blocking NLRP3 inflammasome and IL-1β.

Autophagy suppression potentiates the anti-glioblastoma effect of asparaginase in vitro and in vivo

Asparaginase has been reported to be effective in the treatment of various leukemia and several malignant solid cancers. However, the anti-tumor effect of asparaginase is always restricted due to complicated mechanisms. Herein, we investigated the mechanisms of how glioblastoma resisted asparaginase treatment and reported a novel approach to enhance the anti-glioblastoma effect of asparaginase. We found that asparaginase could induce growth inhibition and caspase-dependent apoptosis in U87MG/U251MG glioblastoma cells. Meanwhile, autophagy was activated as indicated by autophagosomes formation and upregulated expression of LC3-II. Importantly, abolishing autophagy using chloroquine (CQ) and LY294002 enhanced the cytotoxicity and apoptosis induced by asparaginase in U87MG/U251MG cells. Further study proved that Akt/mTOR and Erk signaling pathways participated in autophagy induction, while reactive oxygen species (ROS) served as an intracellular regulator for both cytotoxicity and autophagy in asparaginase-treated U87MG/U251MG cells. Moreover, combination treatment with autophagy inhibitor CQ significantly enhanced anti-glioblastoma efficacy of asparaginase in U87MG cell xenograft model. Taken together, our results demonstrated that inhibition of autophagy potentiated the anti-tumor effect of asparagine depletion on glioblastoma, indicating that targeting autophagy and asparagine could be a potential approach for glioblastoma treatment.Asparaginase has been reported to be effective in the treatment of various leukemia and several malignant solid cancers. However, the anti-tumor effect of asparaginase is always restricted due to complicated mechanisms. Herein, we investigated the mechanisms of how glioblastoma resisted asparaginase treatment and reported a novel approach to enhance the anti-glioblastoma effect of asparaginase. We found that asparaginase could induce growth inhibition and caspase-dependent apoptosis in U87MG/U251MG glioblastoma cells. Meanwhile, autophagy was activated as indicated by autophagosomes formation and upregulated expression of LC3-II. Importantly, abolishing autophagy using chloroquine (CQ) and LY294002 enhanced the cytotoxicity and apoptosis induced by asparaginase in U87MG/U251MG cells. Further study proved that Akt/mTOR and Erk signaling pathways participated in autophagy induction, while reactive oxygen species (ROS) served as an intracellular regulator for both cytotoxicity and autophagy in asparaginase-treated U87MG/U251MG cells. Moreover, combination treatment with autophagy inhibitor CQ significantly enhanced anti-glioblastoma efficacy of asparaginase in U87MG cell xenograft model. Taken together, our results demonstrated that inhibition of autophagy potentiated the anti-tumor effect of asparagine depletion on glioblastoma, indicating that targeting autophagy and asparagine could be a potential approach for glioblastoma treatment.

Activating Autophagy Enhanced the Antitumor Effect of Antibody Drug Conjugates Rituximab-Monomethyl Auristatin E

Background Antibody drug conjugate (ADC) showed potent therapeutic efficacy in several types of cancers. The role of autophagy in antitumor effects of ADC remains unclear. Methods In this study, the ADC, Rituximab-monomethyl auristatin E (MMAE) with a Valine–Citrulline cleavable linker, was designed to investigate its therapeutic efficacy against non-Hodgkin lymphoma (NHL) as well as the underlying mechanisms. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to detect growth inhibition in B-cell lymphoma cell lines, Ramos and Daudi cells, which were treated by Rituximab-MMAE alone or combined with autophagy conditioner. Apoptosis was detected by flow cytometry and immunohistochemistry, and apoptosis inhibitor was employed to discover the relationship between autophagy and apoptosis during the Rituximab-MMAE treatment. Autophagy was determined by three standard techniques which included confocal microscope, transmission electron microscope, and western blots. Ramos xenograft tumors in BALB/c nude mice were established to investigate the antitumor effect of combination use of Rituximab-MMAE and autophagy conditioner in B-NHL therapy. Results Our results showed that Rituximab-MMAE elicited caspase-3-dependent apoptosis in NHL cells and exhibited potent therapeutic efficacy in vivo. Autophagy, which was characterized by upregulated light chain 3-II expression, and accumulation of autophagosomes, was triggered during the Rituximab-MMAE treatment. Meanwhile, inactivation of Akt/mTOR pathway was shown to be involved in the autophagy triggered by Rituximab-MMAE, indicating a probable mechanism of the ADC-initiated autophagy. Importantly, inhibition of autophagy by chloroquine suppressed the Rituximab-MMAE-induced apoptosis, while activating autophagy by rapamycin significantly enhanced the therapeutic effect of Rituximab-MMAE both in vitro and in vivo. Conclusion Our data elucidated the critical relationship between autophagy and apoptosis in Rituximab-MMAE-based therapy and highlighted the potential approach for NHL therapy by combined administration of the ADC and autophagy activator.

In vivo hepatocellular expression of interleukin-22 using penetratin-based hybrid nanoparticles as potential anti-hepatitis therapeutics

Hepatocellular injury is the pathological hallmark of hepatitis and a crucial driver for the progression of liver diseases, while the treatment options are commonly restricted. Interleukin-22 (IL-22) has attracted special attention as a potent survival factor for hepatocytes that both prevents and repairs the injury of hepatocytes through activation of STAT3 signaling pathway. We hypothesized that the ability to generate potent expression of IL-22 locally for the treatment of severe hepatocellular injury in hepatitis was a promising strategy to enhance efficacy and overcome off-target effects. Accordingly, we developed a polypeptide penetratin-based hybrid nanoparticle system (PDPIA) carrying IL-22 gene by a self-assembly process. This nanocomplex modified with penetratin featured direct translocation across the cellular or endosomal membrane but mild zeta-potential to facilitate the high cellular internalization and endosomal escape of the gene cargos as well as scarcely Kupffer cells uptake. More importantly, PDPIA afforded preferential liver accumulation and predominant hepatocytes internalization following systemic administration, which showed pharmacologically suitable organ and sub-organ-selective properties. Subsequent studies confirmed a considerable protective role of PDPIA in a model of severe hepatitis induced by concanavalin A, evidenced by reduced hepatocellular injury and evaded immune response. The locally expressed IL-22 by PDPIA activated STAT3/Erk signal transduction, and thus promoted hepatocyte regeneration, inhibited reactive oxygen species (ROS) accumulation as well as prevented the dysfunction of mitochondrial. In addition, this system did not manifest side effects or systemic toxicity in mice. Collectively, the high versatility of PDPIA rendered its promising applications might be an effective agent to treat various hepatic disorders.

Inhibition of autophagy potentiated the anti-tumor effects of VEGF and CD47 bispecific therapy in glioblastoma

Glioblastoma, characterized by extensive microvascular proliferation and invasive tumor growth, is one of the most common and lethal malignancies in adults. Benefits of the conventional anti-angiogenic therapy were only observed in a subset of patients and limited by diverse relapse mechanism. Fortunately, recent advances in cancer immunotherapy have offered new hope for patients with glioblastoma. Herein, we reported a novel dual-targeting therapy for glioblastoma through simultaneous blockade of VEGF and CD47 signaling. Our results showed that VEGFR1D2-SIRPαD1, a VEGF and CD47 bispecific fusion protein, exerted potent anti-tumor effects via suppressing VEGF-induced angiogenesis and activating macrophage-mediated phagocytosis. Meanwhile, autophagy was activated by VEGFR1D2-SIRPαD1 through inactivating Akt/mTOR and Erk pathways in glioblastoma cells. Importantly, autophagy inhibitor or knockdown of autophagy-related protein 5 potentiated VEGFR1D2-SIRPαD1-induced macrophage phagocytosis and cytotoxicity against glioblastoma cells. Moreover, suppression of autophagy led to increased macrophage infiltration, angiogenesis inhibition, and tumor cell apoptosis triggered by VEGF and CD47 dual-targeting therapy, thus eliciting enhanced anti-tumor effects in glioblastoma. Our data revealed that VEGFR1D2-SIRPαD1 alone or in combination with autophagy inhibitor could effectively elicit potent anti-tumor effects, highlighting potential therapeutic strategies for glioblastoma through disrupting angiogenetic axis and CD47-SIRPα anti-phagocytic axis alone or in combination with autophagy inhibition.