Red Blood Cell-Derived Extracellular Vesicles Display Endogenous Antiviral Effects and Enhance the Efficacy of Antiviral Oligonucleotide Therapy.

The COVID-19 pandemic has resulted in a large number of fatalities and, at present, lacks a readily available curative treatment for patients. Here, we demonstrate that unmodified red blood cell-derived extracellular vesicles (RBCEVs) can inhibit SARS-CoV-2 infection in a phosphatidylserine (PS) dependent manner. Using T cell immunoglobulin mucin domain-1 (TIM-1) as an example, we demonstrate that PS receptors on cells can significantly increase the adsorption and infection of authentic and pseudotyped SARS-CoV-2 viruses. RBCEVs competitively inhibit this interaction and block TIM-1-mediated viral entry into cells. We further extend the therapeutic efficacy of this antiviral treatment by loading antisense oligonucleotides (ASOs) designed to target conserved regions of key SARS-CoV-2 genes into RBCEVs. We establish that ASO-loaded RBCEVs are efficiently taken up by cells in vitro and in vivo to suppress SARS-CoV-2 replication. Our findings indicate that this RBCEV-based SARS-CoV-2 therapeutic displays promise as a potential treatment capable of inhibiting SARS-CoV-2 entry and replication.

Robust delivery of RIG-I agonists using extracellular vesicles for anti-cancer immunotherapy.

The RIG-I pathway can be activated by RNA containing 5' triphosphate, leading to type I interferon release and immune activation. Hence, RIG-I agonists have been used to induce immune responses against cancer as potential immunotherapy. However, delivery of 5' triphosphorylated RNA molecules as RIG-I agonists to tumour cells in vivo is challenging due to the susceptibility of these molecules to degradation. In this study, we demonstrate the use of extracellular vesicles (EVs) from red blood cells (RBCs), which are highly amenable for RNA loading and taken up robustly by cancer cells, for RIG-I agonist delivery. We evaluate the anti-cancer activity of two novel RIG-I agonists, the immunomodulatory RNA (immRNA) with a unique secondary structure for efficient RIG-I activation, and a 5' triphosphorylated antisense oligonucleotide with dual function of RIG-I activation and miR-125b inhibition (3p-125b-ASO). We find that RBCEV-delivered immRNA and 3p-125b-ASO trigger the RIG-I pathway, and induce cell death in both mouse and human breast cancer cells. Furthermore, we observe a significant suppression of tumour growth coupled with increased immune cell infiltration mediated by the activation of RIG-I cascade after multiple intratumoral injections of RBCEVs loaded with immRNA or 3p-125b-ASO. Targeted delivery of immRNA using RBCEVs with EGFR-binding nanobody administrated via intrapulmonary delivery facilitates the accumulation of RBCEVs in metastatic cancer cells, leading to potent tumour-specific CD8+ T cells immune response. This contributes to prominent suppression of breast cancer metastasis in the lung. Hence, this study provides a new strategy for efficient RIG-I agonist delivery using RBCEVs for immunotherapy against cancer and cancer metastasis.

Breast cancer metastasis to brain results in recruitment and activation of microglia through annexin-A1/formyl peptide receptor signaling.

BACKGROUND: Despite advancements in therapies, brain metastasis in patients with triple negative subtype of breast cancer remains a therapeutic challenge. Activated microglia are often observed in close proximity to, or within, malignant tumor masses, suggesting a critical role that microglia play in brain tumor progression. Annexin-A1 (ANXA1), a glucocorticoid-regulated protein with immune-regulatory properties, has been implicated in the growth and metastasis of many cancers. Its role in breast cancer-microglia signaling crosstalk is not known. METHODS: The importance of microglia proliferation and activation in breast cancer to brain metastasis was evaluated in MMTV-Wnt1 spontaneous mammary tumor mice and BALBc mice injected with 4T1 murine breast cancer cells into the carotid artery using flow cytometry. 4T1 induced-proliferation and migration of primary microglia and BV2 microglia cells were evaluated using 2D and coculture transwell assays. The requirement of ANXA1 in these functions was examined using a Crispr/Cas9 deletion mutant of ANXA1 in 4T1 breast cancer cells as well as BV2 microglia. Small molecule inhibition of the ANXA1 receptor FPR1 and FPR2 were also examined. The signaling pathways involved in these interactions were assessed using western blotting. The association between lymph node positive recurrence-free patient survival and distant metastasis-free patient survival and ANXA1 and FPR1 and FPR2 expression was examined using TCGA datasets. RESULTS: Microglia activation is observed prior to brain metastasis in MMTV-Wnt1 mice with primary and secondary metastasis in the periphery. Metastatic 4T1 mammary cancer cells secrete ANXA1 to promote microglial migration, which in turn, enhances tumor cell migration. Silencing of ANXA1 in 4T1 cells by Crispr/Cas9 deletion, or using inhibitors of FPR1 or FPR2 inhibits microglia migration and leads to reduced activation of STAT3. Finally, elevated ANXA1, FPR1 and FPR2 is significantly associated with poor outcome in lymph node positive patients, particularly, for distant metastasis free patient survival. CONCLUSIONS: The present study uncovered a network encompassing autocrine/paracrine ANXA1 signaling between metastatic mammary cancer cells and microglia that drives microglial recruitment and activation. Inhibition of ANXA1 and/or its receptor may be therapeutically rewarding in the treatment of breast cancer and secondary metastasis to the brain.

RNA-Sequencing-Based Transcriptomic Analysis Reveals a Role for Annexin-A1 in Classical and Influenza A Virus-Induced Autophagy.

Influenza viruses have been shown to use autophagy for their survival. However, the proteins and mechanisms involved in the autophagic process triggered by the influenza virus are unclear. Annexin-A1 (ANXA1) is an immunomodulatory protein involved in the regulation of the immune response and Influenza A virus (IAV) replication. In this study, using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR associated protein 9) deletion of ANXA1, combined with the next-generation sequencing, we systematically analyzed the critical role of ANXA1 in IAV infection as well as the detailed processes governing IAV infection, such as macroautophagy. A number of differentially expressed genes were uniquely expressed in influenza A virus-infected A549 parental cells and A549 ∆ANXA1 cells, which were enriched in the immune system and infection-related pathways. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway revealed the role of ANXA1 in autophagy. To validate this, the effect of mechanistic target of rapamycin (mTOR) inhibitors, starvation and influenza infection on autophagy was determined, and our results demonstrate that ANXA1 enhances autophagy induced by conventional autophagy inducers and influenza virus. These results will help us to understand the underlying mechanisms of IAV infection and provide a potential therapeutic target for restricting influenza viral replication and infection.

Annexin-A1 promotes RIG-I-dependent signaling and apoptosis via regulation of the IRF3-IFNAR-STAT1-IFIT1 pathway in A549 lung epithelial cells.

Within the last century, millions of lives have been lost to the four major Influenza pandemics. These influenza pandemics were all caused by Influenza Type A viruses (IAV) through their ability to undergo antigenic drifts and shifts. A greater understanding of IAV and host-pathogen interactions is required to develop effective therapeutics against future outbreaks. Annexin A1 (ANXA1) is a phospholipid binding, calcium-dependent protein known to play essential roles in multiple cellular functions including inflammation, proliferation, migration, and apoptosis. ANXA1 was previously shown to enhance apoptosis after IAV infection. The current study explores the role of ANXA1 in IAV infection of A549 lung epithelial cells further in the context of RIG-I-dependent signaling using A549 and Crispr/Cas9 ANXA1 deleted (A549∆ANXA1) cells. ANXA1 was found to enhance the expression of a cytoplasmic RNA sensor, RIG-I basally and post-infection. RIG-I activation by 5'ppp-RNA in A549 lung epithelial cell induces apoptotic cell death, which is inhibited when ANXA1 is deleted, and reversed when ANXA1 is re-expressed. RIG-I activation by 5'ppp-RNA stimulates the production of IFNß from lung epithelial cells to the same extent as monocytic cells, albeit very late after infection at 48-72 h, through IRF3 and STAT1 activation. ANXA1 deletion delays the phosphorylation of IRF3 and STAT1, leading to lower expression of interferon-stimulated genes, such as IFIT1, and silencing IFIT1 inhibited RIG-I-induced cell death. In all, these results suggest that ANXA1 plays a regulatory role in RIG-I signaling and cell death in A549 lung epithelial cells.

Annexin-A1 - A Blessing or a Curse in Cancer?

Annexin-A1 (ANXA1), a potent endogenous immunomodulatory protein has been implicated in multiple functions essential in cancer, including cell proliferation, apoptosis, chemosensitivity, metastasis, and invasion. ANXA1 expression is varied depending on tumor type, and there are contradictory reports on its role in the regulation of proliferation and tumor growth. Here, we summarize the differing reports on cell proliferation and metastasis and attempt to discuss the reasons behind these different effects. ANXA1 plays a role as a homeostatic protein that regulates essential transcription factors and miRNAs. A more coherent understanding of ANXA1 in cancer could present a more biologically meaningful and clinically relevant strategy.

Annexin-A1 enhances breast cancer growth and migration by promoting alternative macrophage polarization in the tumour microenvironment.

Macrophages are potent immune cells with well-established roles in the response to stress, injury, infection and inflammation. The classically activated macrophages (M1) are induced by lipopolysaccharide (LPS) and express a wide range of pro-inflammatory genes. M2 macrophages are induced by T helper type 2 cytokines such as interleukin-4 (IL4) and express high levels of anti-inflammatory and tissue repair genes. The strong association between macrophages and tumour cells as well as the high incidences of leukocyte infiltration in solid tumours have contributed to the discovery that tumour-associated macrophages (TAMs) are key to tumour progression. Here, we investigated the role of Annexin A1 (ANXA1), a well characterized immunomodulatory protein on macrophage polarization and the interaction between macrophages and breast cancer cells. Our results demonstrate that ANXA1 regulates macrophage polarization and activation. ANXA1 can act dually as an endogenous signalling molecule or as a secreted mediator which acts via its receptor, FPR2, to promote macrophage polarization. Furthermore, ANXA1 deficient mice exhibit reduced tumour growth and enhanced survival in vivo, possibly due to increased M1 macrophages within the tumor microenvironment. These results provide new insights into the molecular mechanisms of macrophage polarization with therapeutic potential to suppress breast cancer growth and metastasis.

Annexin-A1 regulates microRNA-26b* and microRNA-562 to directly target NF-κB and angiogenesis in breast cancer cells.

Annexin 1 (ANXA1) is an endogenous anti-inflammatory protein implicated in cancer. ANXA1 was previously shown to be regulated by hsa-miR-196a. However, whether ANXA1 itself regulates microRNA (miR) expression is unknown. Therefore, we investigated the regulation of miR by ANXA1 in MCF7 breast cancer cells. MCF7-EV (Empty vector) and MCF7-V5 (ANXA1-V5 expressing cells) were subjected to a miR microarray. Microarray analysis revealed a number of miRNAs which were dysregulated in MCF7-V5 cells. 2 novel miRNAs (miR562 and miR26b*) were validated, cloned and functionally characterized. As ANXA1 constitutively activates NF-κB activity to modulate breast cancer metastasis, we found that miR26b* and miR562 directly targeted the canonical NF-κB pathway by targeting the 3' UTR and inhibiting expression of Rel A (p65) and NF-κB1 (p105) respectively. MiR562 inhibited wound healing, which was reversed when ANXA1 was overexpressed. Overexpression of either miR562 or miR26b* in MCF-7 cells enhanced endothelial tube formation when cocultured with human umbilical cord endothelial cells while conversely, treatment of MCF7 cells with either anti-miR562 or anti-miR26b* inhibited endothelial tube formation after co-culture. Further analysis of miR562 revealed that miR562-transfected cell conditioned media enhances endothelial cell tube formation, indicating that miR562 increased angiogenic secreted factors from MCF-7 breast tumor cells. TNFα was increased upon overexpression of miR562, which was reversed when ANXA1 was co-transfected In conclusion, this data suggests that ANXA1-regulated miR26b* and miR562 may play a role in wound healing and tumor-induced endothelial cell tube formation by targeting NF-κB expression and point towards a potential therapeutic target for breast cancer.