G-Quadruplex Linked DNA Guides Selective Transfection into Nucleolin-Overexpressing Cancer Cells.

Gene therapy is a promising approach for treating tumors. Conventional approaches of DNA delivery depending on non-viral or viral vectors are unsatisfactory due to the concerns of biosafety and cell-targeting efficiency. The question how to deliver DNA into tumor cells efficiently and selectively is a major technological problem in tumor gene therapy. Here, we develop a vector-free gene transfer strategy to deliver genes effectively and selectively by taking advantage of targeting nucleolin. Nucleolin, a shuttle protein moving between cell membrane, cytoplasm and nuclei, is overexpressed in tumor cells. It has a natural ligand G-quadruplex (Gq). Gq-linked DNA (Gq-DNA) is likely to be internalized by ligand dependent uptake mechanisms independently of vectors after neutralizing negative charges of cell membrane by targeting nucleolin. This strategy is referred to as Gq-DNA transfection. Benefiting from its high affinity to nucleolin, Gq-DNA can be effectively delivered into nucleolin-positive tumor cells even nuclei. Gq-DNA transfection is characterized by low cytotoxicity, high efficiency, ease of synthesis, high stability in serum, direct access into nuclei, and specific nucleolin-positive tumor cell targeting.

Crosstalk between macrophage-derived PGE2 and tumor UHRF1 drives hepatocellular carcinoma progression.

Background: Tumor-associated macrophages (TAMs) and dysregulated tumor epigenetics contribute to hepatocellular carcinoma (HCC) progression. However, the mechanistic interactions between TAMs and tumor epigenetics remain poorly understood. Methods: Immunohistochemistry and multiplexed fluorescence staining were performed to evaluate the correlation between TAMs numbers and UHRF1 expression in human HCC tissues. PGE2 neutralizing antibody and COX-2 inhibitor were used to analyze the regulation of TAMs isolated from HCC tissues on UHRF1 expression. Multiple microRNA prediction programs were employed to identify microRNAs that target UHRF1 3'UTR. Luciferase reporter assay was applied to evaluate the regulation of miR-520d on UHRF1 expression. Chromatin immunoprecipitation (ChIP) assays were performed to assess the abundance of H3K9me2 in the KLF6 promoter and DNMT1 in the CSF1 promoter regulated by UHRF1. The functional roles of TAM-mediated oncogenic network in HCC progression were verified by in vitro colony formation assays, in vivo xenograft experiments and analysis of clinical samples. Results: Here, we find that TAMs induce and maintain high levels of HCC UHRF1, an oncogenic epigenetic regulator. Mechanistically, TAM-derived PGE2 stimulates UHRF1 expression by repressing miR-520d that targets the 3'-UTR of UHRF1 mRNA. In consequence, upregulated UHRF1 methylates H3K9 to diminish tumor KLF6 expression, a tumor inhibitory transcriptional factor that directly transcribes miR-520d. PGE2 reduces KLF6 occupancy in the promoter of miR-520d, dampens miR-520d expression, and sustains robust UHRF1 expression. Moreover, UHRF1 promotes CSF1 expression by inducing DNA hypomethylation of the CSF1 promoter and supports TAM accumulation. Conclusions: Capitalizing on studies on HCC cells and tissues, animal models, and clinical information, we reveal a previously unappreciated TAM-mediated oncogenic network via multiple reciprocal enforcing molecular nodes. Targeting this network may be an approach to treat HCC patients.

IDO-inhibitor potentiated immunogenic chemotherapy abolishes primary tumor growth and eradicates metastatic lesions by targeting distinct compartments within tumor microenvironment.

Immunogenic chemotherapy (IC) is a type of chemotherapy where certain chemodrugs induce immunogenic cancer cell death (ICD), which in turn arouses T cell antitumor immunity. However, IC concurrently upregulates a key immune suppressor, indoleamine-2,3-dioxygenase (IDO), in both cancer cells and immune cells. IDO-mediated immunosuppression significantly offsets IC's therapeutic benefits in cancer patients, suggesting a necessity of combination with IDO inhibitors. Here, we report an enzyme-, pH-, and redox-triple-sensitive nanosystem using mesoporous silica nanoparticles (MSNs) as a core encapsulating doxorubicin (DOX, an immunogenic chemodrug); the core is coated with a shell (ß-CD-PEI/Ge1MT) for co-delivering 1-methyl-D-tryptophan (1 MT, an IDO inhibitor). By using these responsivenesses sequentially triggering the release of 1 MT into tumor extracellular compartment and DOX into intracellular endo/lysosomal compartment, this nanosystem (DOX@GMTMSNs) precisely delivers the drugs to their target cells residing in different compartments. Released 1 MT uptake by IDO-expressing dendritic cells (DCs) and cancer cells suppresses IDO activity, reducing immunosuppressive Tregs' presence; DOX unloaded within cancer cells induces ICD, promoting effector T-cell infiltration. In two preclinical cancer models, DOX@GMTMSNs potentiate both tumor local and systemic antitumor immunity, suppressing primary tumor growth by 78% with an 83% reduction in metastatic foci, as well as extending animal survival, thus strongly demonstrating DOX@ GMTMSNs' clinical translational potential.

Targeting Inhibition of Foxp3 by MMP2/9 Sensitive Short Peptide Linked P60 Fusion Protein 6(P60-MMPs) to Enhance Antitumor Immunity.

Regulatory T-cells (Tregs) play an important role in tumor immunosuppressive network, thus Tregs-targeted strategy is expected to enhance antitumor immunity and improve the effect of immunotherapy. Short peptide P60 can bind to the forkhead box protein P3 (Foxp3), a crucial transcriptional regulator for the development and inhibitory function of Tregs, and inhibit Foxp3 nuclear translocation in Tregs. However, its treatment effect in cancer is limited due to nonspecificity. Therefore, realizing the specific delivery of P60 in tumor microenvironment will greatly facilitate its Treg-suppressing effect for tumor therapeutics. Herein, utilizing the unique matrix metallase protease 2/9 (MMP2/9) overexpressing feature in tumor tissues, a fusion protein 6(P60-MMPs) containing six segments of P60 linked by MMP2/9-sensitive peptides is constructed for antitumor targeting immunotherapy. The fusion protein 6(P60-MMPs) specifically degrades into short peptide P60 in tumor, and then binds to Foxp3 to inhibit Foxp3 nuclear translocation in Tregs, thus impairing Tregs' activity. This fusion protein efficiently inhibits murine breast cancer 4T1 transplanted tumor growth and decreases lung metastasis through down-regulating tumor-infiltrated Tregs and up-regulating CD8+ T cells in tumor tissue. The study develops a Treg-targeted anticancer fusion protein with effective therapeutic activity, suggesting its potential in clinical translation.

Uhrf1-Mediated Tnf-α Gene Methylation Controls Proinflammatory Macrophages in Experimental Colitis Resembling Inflammatory Bowel Disease.

Macrophages drive the pathological process of inflammatory bowel diseases (IBD) mostly by secreting proinflammatory cytokines, such as Tnf-α. Recent studies have indicated the association between epigenetic modifications and macrophage functions. However, epigenetic mechanisms regulating macrophages' functional involvement in IBD remain unknown. In this study, we investigated whether the epigenetic regulator Uhrf1 plays a role in innate immunity by functionally regulating macrophages in intestines. We employed two transgenic strains of mice (one with Uhrf1 deficiency in macrophages [Uhrf1fl/flLyz2-Cre mice] and the other with the two mutations at Uhrf1's DNA methylation regulatory site [Uhrf1YP187/188AA mice]) to assess their susceptibility to dextran sodium sulfate-induced colitis. We examined the cytokines derived from Uhrf1fl/flLyz2-Cre and Uhrf1YP187/188AA macrophages in response to LPS stimulation. We also analyzed the effects of proinflammatory cytokines on Uhrf1 expression in macrophages. The data demonstrated that Uhrf1 deficiency and Uhrf1YP187/188AA mutation resulted in severe colitis in the dextran sodium sulfate-treated mice. In vitro analysis revealed the hypomethylation of Tnf-α promoter and the increased Tnf-α expression in Uhrf1fl/flLyz2-Cre and Uhrf1YP187/188AA macrophages in response to LPS stimulation, and anti-Tnf-α therapy implied the key role of Tnf-α to the aggravated colitis in Uhrf1-deficient mice. Exogenous Tnf-α destabilized Uhrf1 protein through ubiquitination-mediated protein degradation, triggering macrophage activation. In conclusion, we identified that Uhrf1-mediated DNA methylation controls Tnf-α expression of macrophages in the experimental colitis resembling IBD. The epigenetic mechanisms that activate macrophages may provide new therapeutic targets for IBD treatment.

Functional extracellular vesicles engineered with lipid-grafted hyaluronic acid effectively reverse cancer drug resistance.

Multidrug resistance (MDR) is a key issue accounting for ineffectiveness of cancer chemotherapy. Numerous multifunctional nanocarriers have been developed to increase drug delivery efficacy and inhibit drug efflux for overcoming cancer drug resistance. However, limited success has been achieved in clinic because of nanocarriers' complicated multi-step fabrication procedures and their undesired side toxicity as well as potential immunogenicity. Here, hyaluronic acid (HA) functionalized extracellular vesicles (EVs) are generated as natural vehicles to efficiently deliver doxorubicin (DOX) and reverse MDR. The EVs isolated from noncancerous HEK293T cells (hEVs) reduce P-glycoprotein (P-gp) expression in drug resistant MCF7/ADR cells. To acquire tumor-targeting capability, hEVs are modified with lipidomimetic chains-grafted HA (lipHA) by a simple incubation. Owing to CD44-mediated cancer-specific targeting and P-gp suppressive capability, the HA-functionalized hEVs (lipHA-hEVs) remarkably promote the intracellular DOX accumulation in drug resistant breast cancer cells. In preclinical MDR tumor models, lipHA-hEVs deeply penetrate into tumor tissue and effectively transport DOX into tumor local, while eliminating DOX's systemic toxicity. Importantly, DOX@lipHA-hEVs inhibited MDR tumor growth by 89% and extend animal survival time by approximately 50%. Thus, our engineered tumor-targeting hEVs are promising natural carriers for overcoming cancer MDR.