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Exosomes are small extracellular vehicles which could transport genetic materials and proteins between cells. Although there are reports about exosomes crossing the blood-brain barrier (BBB), the underlying mechanisms still need further study. We found that exosomes from primary brain tumors could upregulate the expression of Lipocalin-2 (LCN2) in bEnd.3 brain microvascular endothelial cells (BMVECs). Furthermore, exosomes increased the membrane fluidity of bEnd.3 cells in an LCN2 dependent manner. Both intraperitoneal injection and caudal vein injection of LCN2 increased the number of nanocapsules crossing the BBB. Evans Blue staining revealed that LCN2 does not interrupt the integrity of the BBB, as observed in the traumatic brain injury model. Tandem mass tags quantitative proteomics and bioinformatics analysis revealed that LCN2 is upregulated by exosomes via the JAK-STAT3 pathway, but not delivered from exosomes. Knocking down LCN2 in bEnd.3 cells significantly abrogated the effect of exosomes on BMVEC membrane fluidity. Previously, we have reported that 2-methacryloyloxyethyl phosphorylcholine (MPC) and a peptide crosslinker could encapsulate mAbs to achieve nanocapsules. The nanocapsules containing choline analogs could effectively penetrate the BBB to deliver therapeutic monoclonal antibodies (tAbs) to the glioma. However, the delivered tAbs could be significantly reduced by blocking the release of exosomes from the gliomas. Application of tAb nanocapsules prior to treatment with MK2206, an AKT pathway inhibitor that has been shown to inhibit the production of exosomes, resulted in a better combination. Insights from this study provide a mechanistic framework with regard to how glioblastomas hijack BMVECs using exosomes. In addition, we provide a strategy for maximizing the effect of the choline-containing nanocapsules and MK2206 combination. These results also demonstrate the therapeutic role of tAbs in glioblastoma and brain tumor metastasis, by shedding new light on strategies that can be used for BBB-penetrating therapies.

Background Targeting glioblastoma (GBM) energy metabolism through multiple metabolic pathways has emerged as an effective therapeutic approach. Dual inhibition of phospholipid and mitochondrial metabolism with cytoplasmic phospholipase A2 (cPLA2) knockdown and metformin treatment could be a potential strategy. However, the strategic prerequisite is to explore a carrier capable of co-delivering the therapeutic combination to cross the blood-brain barrier (BBB) and preferentially accumulate at the GBM site. Methods Blood exosomes (Exos) were selected as the combination delivery carriers. The cellular uptake of Exos and the therapeutic effects of the combination strategy were evaluated in primary GBM cells. In vivo GBM-targeted delivery efficiency and anti-GBM efficacy were tested in a patient-derived xenograft (PDX) model. Results Here, we showed that the Exos-mediated cPLA2 siRNA/metformin combined strategy could regulate GBM energy metabolism for personalized treatment. Genomic analysis and experiments showed that polymerase 1 and transcript release factor (PTRF, a biomarker of GBM) positively regulated the uptake of Exos by GBM cells, confirming the feasibility of the delivery strategy. Further, Exos could co-load cPLA2 siRNA (sicPLA2) and metformin and co-deliver them across the BBB and into GBM tissue. The mitochondrial energy metabolism of GBM was impaired with this combination treatment (Exos-Met/sicPLA2). In the PDX GBM model, systemic administration of Exos-Met/sicPLA2 reduced tumor growth and prolonged survival. Conclusions Our findings demonstrated that Exos-based combined delivery of sicPLA2 and metformin selectively targeted the GBM energy metabolism to achieve antitumor effects, showing its potential as a personalized therapy for GBM patients.

Background Metabolism remodeling is a hallmark of glioblastoma (GBM) that regulates tumor proliferation and the immune microenvironment. Previous studies have reported that increased polymerase 1 and transcript release factor (PTRF) levels are associated with a worse prognosis in glioma patients. However, the biological role and the molecular mechanism of PTRF in GBM metabolism remain unclear. Methods The relationship between PTRF and lipid metabolism in GBM was detected by nontargeted metabolomics profiling and subsequent lipidomics analysis. Western blotting, quantitative real-time PCR, and immunoprecipitation were conducted to explore the molecular mechanism of PTRF in lipid metabolism. A sequence of in vitro and in vivo experiments (both xenograft tumor and intracranial tumor mouse models) were used to detect the tumor-specific impacts of PTRF. Results Here, we show that PTRF triggers a cytoplasmic phospholipase A2 (cPLA2)–mediated phospholipid remodeling pathway that promotes GBM tumor proliferation and suppresses tumor immune responses. Research in primary cell lines from GBM patients revealed that cells overexpressing PTRF show increased cPLA2 activity—resulting from increased protein stability—and exhibit remodeled phospholipid composition. Subsequent experiments revealed that PTRF overexpression alters the endocytosis capacity and energy metabolism of GBM cells. Finally, in GBM xenograft and intracranial tumor mouse models, we showed that inhibiting cPLA2 activity blocks tumor proliferation and prevents PTRF-induced reduction in CD8+ tumor-infiltrating lymphocytes. Conclusions The PTRF-cPLA2 lipid remodeling pathway promotes tumor proliferation and suppresses immune responses in GBM. In addition, our findings highlight multiple new therapeutic targets for GBM.

Liposomes have been developed as drug delivery carriers to enhance the antitumor efficiency of therapeutic agents. Lipusu® (Lip), a paclitaxel (PTX) liposome, has been widely used in the treatment of breast cancer. Compared with PTX, Lip could change the biodistribution and reduce the systemic toxicity. However, there was no positive effect on the entry of PTX into tumor cells, and thus the therapeutic effect was not significantly improved. Therefore, it is meaningful to engineer Lip for improving tumor cellular uptake efficiency. Here, lysophosphatidylcholine (LPC)-engineered Lip (LPC-Lip) was constructed

Immunosuppression is a key factor leading to a low therapeutic efficiency of the currently used immunotherapies. Monotherapies are unable to overcome immunosuppression because of the complex interplay of immune cells in tumors. Herein, we report a multifunctional nanomodulator (MFNM) as a carrier to deliver different types of immune modulators for comodulating multiple pathways. An MFNM has a core-shell structure, in which small-molecule drugs are encapsulated in a mesoporous silica nanoparticle (MSN) core with a pH-responsive polymer layer. Further, the polymeric shell provides active sites that are readily modifiable by multiple types of antibodies to regulate the immune-related processes. By codelivering cyclophosphamide (CTX), αPD-L1 (B7-H1), and α4-1BB (CD137L) monoclonal antibodies (mAbs) to tumors, an MFNM has been shown to regulate multiple immune pathways and enhance an antitumor immunity. As antibodies and small-molecule drugs loaded in an MFNM can be modified based on the tumor type, the MFNM provides a feasible platform for the development of advanced immunotherapies that require simultaneous modulation of multiple biological processes.

Rationale: Developing an effective nanoplatform to realize 'multi-in-one' is essential to broaden the therapeutic potential of combination therapy. Exosomes are ideal candidates since their intrinsic abilities of integrating multiple contents and functions. However, only limited efforts have been devoted to engineering exosomes to integrate the needed properties, also considering the safety and yield, for tumor-targeted and efficient gene/chemo combination therapy. Methods: Herein, by manipulating the exosome membrane, blood exosomes with high abundance and safety are engineered as a versatile combinatorial delivery system, where the doxorubicin (Dox) and cholesterol-modified miRNA21 inhibitor (miR-21i) are co-embedded into the lipid bilayer of exosomes, and the magnetic molecules and endosomolytic peptides L17E are bind to the exosome membrane through ligand-receptor coupling and electrostatic interactions, respectively. Results: It is proved that such engineering strategy not only preserves their intrinsic features, but also readily integrates multiple properties of tumor targeting, efficient transfection and gene/chemo combination therapy into blood exosomes. The lipid bilayer structure of exosomes allows them to co-load Dox and miR-21i with high-payloads. Moreover, profiting from the integration of magnetic molecules and L17E peptides, the engineered exosomes exhibit an enhanced tumor accumulation and an improved endosome escape ability, thereby specifically and efficiently delivering encapsulated cargos to tumor cells. As a result, a remarkable inhibition of tumor growth is observed in the tumor-bearing mice, and without noticeable side effects. Conclusions: This study demonstrates the potential of engineered blood exosomes as feasible co-delivery nanosystem for tumor-targeted and efficient combination therapy. Further development by replacing the drugs combined regimens can potentially make this engineered exosome become a general platform for the design of safe and effective combination therapy modality.

Genomic instability (GI) drives tumor heterogeneity and promotes tumor progression and therapy resistance. However, causative factors underlying GI and means for clinical detection of GI in glioma are inadequately identified. We describe here that elevated expression of a gene module coexpressed with CDC20 (CDC20-M), the activator of the anaphase-promoting complex in the cell cycle, marks GI in glioma. The CDC20-M, containing 139 members involved in cell proliferation, DNA damage response, and chromosome segregation, was found to be consistently coexpressed in glioma transcriptomes. The coexpression of these genes was conserved across multiple species and organ systems, particularly in human neural stem and progenitor cells. CDC20-M expression was not correlated with the morphological subtypes, nor with the recently defined molecular subtypes of glioma. CDC20-M signature was an independent and robust predictor for poorer prognosis in over 1,000 patients from four large databases. Elevated CDC20-M signature enabled the identification of individual glioma samples with severe chromosome instability and mutation burden and of primary glioma cell lines with extensive mitotic errors leading to chromosome mis-segregation. AURKA, a core member of CDC20-M, was amplified in one-third of CDC20-M-high gliomas with gene-dosage-dependent expression. MLN8237, a Food and Drug Administration-approved AURKA inhibitor, selectively killed temozolomide-resistant primary glioma cells in vitro and prolonged the survival of a patient-derived xenograft mouse model with a high-CDC20-M signature. Our findings suggest that application of the CDC20-M signature may permit more selective use of adjuvant therapies for glioma patients and that dysregulated CDC20-M members may provide a therapeutic vulnerability in glioma.

Over 20% of cancer 'driver' genes encode chromatin regulators. Long noncoding RNAs (lincRNAs), which are dysregulated in various cancers, play a critical role in chromatin dynamics and gene regulation by interacting with key epigenetic regulators. It has been previously reported that the lincRNA HOTAIR mediates recruitment of polycomb repressive complex 2 (PRC2) leading to aberrant transcriptional silencing of tumor suppressor genes in glioma and breast cancer. Thus, lincRNA HOTAIR can serve as a promising therapeutic target. Herein, we identified a small-molecule compound AC1Q3QWB (AQB) as a selective and efficient disruptor of HOTAIR-EZH2 interaction, resulting in blocking of PRC2 recruitment and increasing tumor suppressors expression. Methods: Molecular docking and high-throughput screening were performed to identify the small compound, AQB. RIP and ChIRP assays were carried to assess the selective interference of AQB with the HOTAIR-EZH2 interaction. The effects of AQB on tumor malignancy were evaluated in a variety of cancer cell lines and orthotopic breast cancer models. The combination therapy of AQB and 3-Deazaneplanocin A (DZNep), an inhibitor of the histone methyltransferase EZH2 was used in vitro and in orthotopic breast cancer and glioblastoma patient-derived xenograft (PDX) models. Results: Tumor cells highly expressing HOTAIR and EZH2 were sensitive to AQB. APC2, as one of the target genes, was significantly up-regulated by AQB and led to degradation of β-catenin resulting in suppression of Wnt/β-catenin signaling which may contribute to inhibition of tumor growth and metastasis in vitro and in orthotopic breast cancer models. Remarkably, AQB enhanced the toxicity of DZNep in vitro. In orthotopic breast cancer and glioblastoma patient-derived xenografts (PDX) models, the combination of low doses of AQB and DZNep realized much better killing than DZNep treatment alone. Conclusion: AQB is a HOTAIR-EZH2 inhibitor, which blocks PRC2 recruitment and has great potential as an effective agent for targeted cancer therapy.

Exosomes derived from non-tumor cells hold great potential as drug delivery vehicles because of their good biosafety and natural transference of bioactive cargo between cells. However, compared to tumor-derived exosomes, efficient delivery is limited by their weak interactions with tumor cells. It is essential to engineer exosomes that improve tumor cellular internalization efficiency. A simple and effective strategy to enhance tumor cell uptake by engineering the exosome membrane lipids can be established by drawing on the role of lipids in tumor exosomes interacting with tumor cells. Amphiphilic phosphatidylcholine (PC) molecules are inserted into the membrane lipid layer of reticulocyte-derived exosomes (Exos) by simple incubation to construct PC-engineered exosomes (PC-Exos). It is demonstrated that PC-Exos showed significantly enhanced tumor cell internalization and uptake rate compared to native Exos, up to a twofold increase. After therapeutic agent loading, PC-Exos remarkably promotes intracellular drug or RNA accumulation in cancer cells, thus showing enhanced in vitro anti-tumor activity. This work demonstrates the crucial role of engineering exosomal lipids in modulating cancer cellular uptake, which may shed light on the design of high-efficiency exosome-based drug delivery carriers.

Glioblastoma (GBM) is the most common primary central nervous system tumor and has a poor prognosis, with a median survival time of only 14 months from diagnosis. Abnormally expressed long noncoding RNAs (lncRNAs) are important epigenetic regulators of chromatin modification and gene expression regulation in tumors, including GBM. We previously showed that the lncRNA HOTAIR is related to the cell cycle progression and can be used as an independent predictor in GBM. Lysine-specific demethylase 1 (LSD1), binding to 3' domain of HOTAIR, specifically removes mono- and di-methyl marks from H3 lysine 4 (H3K4) and plays key roles during carcinogenesis. In this study, we combined a HOTAIR-EZH2 disrupting agent and an LSD1 inhibitor, AC1Q3QWB (AQB) and GSK-LSD1, respectively, to block the two functional domains of HOTAIR and potentially provide therapeutic benefit in the treatment of GBM. Using an Agilent Human ceRNA Microarray, we identified tumor suppressor genes upregulated by AQB and GSK-LSD1, followed by Chromatin immunoprecipitation (ChIP) assays to explore the epigenetic mechanisms of genes activation. Microarray analysis showed that AQB and GSK-LSD1 regulate cell cycle processes and induces apoptosis in GBM cell lines. Furthermore, we found that the combination of AQB and GSK-LSD1 showed a powerful effect of inhibiting cell cycle processes by targeting CDKN1A, whereas apoptosis promoting effects of combination therapy were mediated by BBC3 in vitro. ChIP assays revealed that GSK-LSD1 and AQB regulate P21 and PUMA, respectively via upregulating H3K4me2 and downregulating H3K27me3. Combination therapy with AQB and GSK-LSD1 on tumor malignancy in vitro and GBM patient-derived xenograft (PDX) models shows enhanced anti-tumor efficacy and appears to be a promising new strategy for GBM treatment through its effects on epigenetic regulation.

Autophagy of mitochondria, termed mitophagy, plays an important role in cerebral ischemia-reperfusion (IR) injury, but the mechanism is not yet clear. Tissue-type plasminogen activator (tPA) is the most important thrombolytic drug in the clinical treatment of ischemic stroke and has neuroprotective effects. Here, we explored the effects of tPA on neuronal apoptosis and mitophagy following IR. We found that knocking out the tPA gene significantly aggravated brain injury and increased neuronal apoptosis and mitochondrial damage. Exposure of neurons to tPA reduced injury severity and protected mitochondria. Further studies demonstrated that this protective effect of tPA was achieved via regulation of FUNDC1-mediated mitophagy. Furthermore, we found that tPA enhanced the expression level of FUNDC1 by activating the phosphorylation of AMPK. In summary, our results confirm that tPA exerts neuroprotective effects by increasing the phosphorylation of AMPK and the expression of FUNDC1, thereby inhibiting apoptosis and improving mitochondrial function., Graphical abstract Image 1, Highlights • After cerebral ischemia, tPA released by neurons has a neuroprotective effect. • tPA modulates mitophagy to decrease oxidative stress and inhibit apoptosis. • The mitochondrial membrane protein FUNDC1 and the AMPK signaling pathway are involved in the neuroprotective process of tPA.

Background The EGFR-vIII mutation is the most common malignant event in GBM. Epigenetic reprogramming in EGFR-activated GBM has recently been suggested to downregulate the expression of tumour suppressor genes. Histone acetylation is important for chromatin structure and function. However, the role and biological function of H2AZK4/7AC in tumours have not yet been clarified. Results In our study, we found that EGFR-vIII negatively regulated H2AZK4/7AC expression though the PI3K/AKT-HDAC2 axis. Because HDAC1 and HDAC2 are highly homologous enzymes that usually form multi-protein complexes for transcriptional regulation and epigenetic landscaping, we simultaneously knocked out HDAC1 and HDAC2 and found that H2AZK4/7AC and H3K27AC were upregulated, which partially released EGFR-vIII-mediated inhibition of USP11, negative regulator of cell cycle. In addition, we demonstrated in vitro and in vivo that FK228 induced G1/S transition arrest in GBM with EGFR-vIII mutation. FK228 could enhance anti-tumour activity by upregulating expression of the tumour suppressor USP11 in GBM cells. Conclusions EGFR-vIII mutation downregulates H2AZK4/7AC and H3K27AC, inhibiting USP11 expression though the PI3K/AKT-HDAC1/2 axis. FK228 is an effective and promising treatment for GBM with EGFR-vIII mutation.

Runt-Related Transcription Factor 1 (RUNX1) is highly expressed in the Mesenchymal (Mes) subtype of glioblastoma (GBM). However, the specific molecular mechanism of RUNX1 in Mes GBM remains largely elusive. In this study, cell and tumor tissue typing were performed by RNA-sequencing. Co-immunoprecipitation (co-IP) and immunofluorescence (IF) were employed to identify members of the RUNX1 transcriptional protein complex. Bioinformatics analysis, chromatin immunoprecipitation (ChIP), and luciferase reporter experiments were utilized to verify target genes. Analyses of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) verified the expression levels and prognoses associated with RUNX1/p-SMAD3/SUV39H1 target genes. In vivo patient-derived xenograft (PDX) studies and in vitro functional studies verified the impact of RUNX1 on the occurrence and development of GBM. The results showed that RUNX1 was upregulated in Mes GBM cell lines, tissues and patients and promoted proliferation and invasion in GBM in a TGFβ pathway-dependent manner in vivo and in vitro. We found and verified that BCL3 and MGP are transcriptionally activated by p-SMAD3 /RUNX1, while MXI1 is transcriptionally suppressed by the RUNX1/SUV39H1-H3K9me3 axis. This finding offers a theoretical rationale for using molecular markers and choosing therapeutic targets for the Mes type of GBM.

Rationale: Competitive endogenous RNA (ceRNA) networks play important roles in posttranscriptional regulation. Their dysregulation is common in cancer. However, ceRNA signatures have been poorly examined in the invasive and aggressive phenotypes of mesenchymal glioblastoma (GBM). This study aims to characterize mesenchymal glioblastoma at the mRNA-miRNA level and identify the mRNAs in ceRNA networks (micNET) markers and their mechanisms in tumorigenesis. Methods: The mRNAs in ceRNA networks (micNETs) of glioblastoma were investigated by constructing a GBM ceRNA network followed by integration with a STRING protein interaction network. The prognostic micNET markers of mesenchymal GBM were identified and validated across multiple datasets. ceRNA interactions were identified between micNETs and miR181 family members. LY2109761, an inhibitor of TGFBR2, demonstrated tumor-suppressive effects on both primary cultured cells and a patient-derived xenograft intracranial model. Results: We characterized mesenchymal glioblastoma at the mRNA-miRNA level and reported a ceRNA network that could separate the mesenchymal subtype from other subtypes. Six genes (TGFBR2, RUNX1, PPARG, ACSL1, GIT2 and RAP1B) that interacted with each other in both a ceRNA-related manner and in terms of their protein functions were identified as markers of the mesenchymal subtype. The coding sequence (CDS) and 3'-untranslated region (UTR) of TGFBR2 upregulated the expression of these genes, whereas TGFBR2 inhibition by siRNA or miR-181a/d suppressed their expression levels. Furthermore, mesenchymal subtype-related genes and the invasion phenotype could be reversed by suppressing the six mesenchymal marker genes. Conclusions: This study suggests that the micNETs may have translational significance in the diagnosis of mesenchymal GBM and may be novel therapeutic targets.

The epidermal growth factor receptor (EGFR) frequently undergoes high-level genomic amplification and variant III (vIII) deletion in adult glioblastoma. MicroRNAs (miRNAs) are recognized to participate in gene expression regulation. We found that miR-524-3p and miR-524-5p were suppressed in the classical molecular subtype of glioblastoma (GBM) from Chinese Glioma Genome Atlas (CGGA) data, and the suppression was associated with EGFR overexpression and EGFRvIII mutation. These two miRNAs improved overall survival time of patients with glioma, and their overexpression could restrain glioma cell migration, proliferation, and cell cycle, and control tumor formation in vivo. Interestingly, both of the miRNAs had a synergistic inhibitory effect on glioma cells. Furthermore, we confirmed that EGFR amplification/EGFRvIII mutation can repress the expression of Pri-miR-524 by histone modification. MiR-524-3p and miR-524-5p inhibited TGF/β, Notch and the Hippo pathway by targeting Smad2, Hes1 and Tead1, respectively; these pathways repressed their common downstream transcription factor, C-myc. More interestingly, C-myc bound to the promoter region of EGFR/EGFRvIII and activated its expression. These findings indicate that miR-524 mediates the EGFR/EGFRvIII stimulating effect. It may serve as a potential therapeutic agent and classical-specific biomarker for the development of glioma.

Worldwide, glioblastoma (GBM) is the most lethal and frequent intracranial tumor. Despite decades of study, the overall survival of GBM patients remains unchanged. epidermal growth factor receptor (EGFR) amplification and gene mutation are thought to be negatively correlated with prognosis. In this study, we used proteomics to determine that UBXN1 is a negative downstream regulator of the EGFR mutation vIII (EGFRvIII). Via bioinformatics analysis, we found that UBXN1 is a factor that can improve glioma patients' overall survival time. We also determined that the down-regulation of UBXN1 is mediated by the upregulation of H3K27me3 in the presence of EGFRvIII. Because NF-κB can be negatively regulated by UBXN1, we believe that EGFRwt/vIII activates NF-κB by suppressing UBXN1 expression. Importantly, we used the latest genomic editing tool, CRISPR/Cas9, to knockout EGFRwt/vIII on exon 17 and further proved that UBXN1 is negatively regulated by EGFRwt/vIII. Furthermore, knockout of EGFR/EGFRvIII could benefit GBM in vitro and in vivo, indicating that CRISPR/Cas9 is a promising therapeutic strategy for both EGFR amplification and EGFR mutation-bearing patients.

In article number https://doi.org/10.1002/advs.201901299, Chunsheng Kang and co‐workers demonstrate the collateral effect of the CRISPR‐Cas13a system in glioma cells. EGFRvIII is a unique EGFR mutant subtype in glioma, and the CRISPR‐Cas13a system induces death in EGFRvIII‐overexpressing glioma cells. Furthermore, CRISPR‐Cas13a inhibits the formation of glioma intracranial tumors in mice. The results indicate the powerful tumor eliminating potential of this system.

Simple Summary The current understanding of miRNA biology is greatly derived from studies on the guide strands and the passenger strands, also called miRNAs*, which are considered as carriers with no sense for long periods. As such, various studies alter the expression of guide strands by manipulating the expression of their primary transcripts or precursors, both of which are premature miRNAs. In this situation, the regulatory miRNA* species may interfere with the phenotypic interpretation against the target miRNA. However, such methods could manipulate the expression of two functionally synergistic miRNAs of the same precursor, leading to therapeutic potential against various diseases, including cancers. Premature miRNAs represent an underappreciated target reservoir and provide molecular targets for “one-two punch” against cancers. Examples of targetable miRNA precursors and available targeting strategies are provided in this review. Abstract Up-to-date knowledge regarding the biogenesis and functioning of microRNAs (miRNAs) has provided a much more comprehensive and concrete view of miRNA biology than anyone ever expected. Diverse genetic origins and biogenesis pathways leading to functional miRNAs converge on the synthesis of ≈21-nucleotide RNA duplex, almost all of which are processed from long premature sequences in a DICER- and/or DROSHA-dependent manner. Formerly, it was assumed that one mature strand of the duplex is preferentially selected for entry into the silencing complex, and the paired passenger strands (miRNA*) are subjected to degradation. However, given the consolidated evidence of substantial regulatory activity of miRNA* species, currently, this preconception has been overturned. Here, we see the caveat and opportunity toward exogenously manipulating the expression of premature miRNA, leading to simultaneous upregulation or downregulation of dual regulatory strands due to altered expressions. The caveat is the overlooked miRNA* interference while manipulating the expression of a target miRNA at the premature stage, wherein lies the opportunity. If the dual strands of a pre-miRNA function synergistically, the overlooked miRNA* interference may inversely optimize the therapeutic performance. Insightfully, targeting the premature miRNAs may serve as the “one-two punch” against diseases, especially cancers, and this has been discussed in detail in this review.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) system holds great promise for the cancer gene therapy. However, due to complicated signal networks and various compensatory mechanisms in tumors, adjusting a single molecular pathway has limited effects on cancer treatments. Herein, a virus-like nanoparticle (VLN) was reported as a versatile nanoplatform to co-deliver CRISPR/Cas9 system and small molecule drugs for effective malignant cancer treatment. VLN has a core-shell structure, in which small molecule drugs and CRISPR/Cas9 system are loaded in the mesoporous silica nanoparticle (MSN)-based core, which is further encapsulated with a lipid shell. This structure allows VLN maintaining stable during blood circulation. As reaching tumors, VLN releases the CRISPR/Cas9 system and small molecule drugs in response to the reductive microenvironment, resulting in the synergistic regulation of multiple cancer-associated pathways. By loading a single guide RNA (sgRNA) targeting programmed death-ligand 1 and axitinib, VLN achieved to disrupt multiple immunosuppressive pathways and suppress the growth of melanoma in vivo. More importantly, VLN can co-deliver almost any combination of sgRNAs and small molecule drugs to tumors, suggesting the great potential of VLN as a general platform for the development of advanced combination therapies against malignant tumors.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) enzyme, Cas13a, holds great promise in cancer treatment due to its potential for selective destruction of tumor cells via collateral effects after target recognition. However, these collateral effects do not specifically target tumor cells and may cause safety issues when administered systemically. Herein, a dual-locking nanoparticle (DLNP) that can restrict CRISPR/Cas13a activation to tumor tissues is described. DLNP has a core-shell structure, in which the CRISPR/Cas13a system (plasmid DNA, pDNA) is encapsulated inside the core with a dual-responsive polymer layer. This polymer layer endows the DLNP with enhanced stability during blood circulation or in normal tissues and facilitates cellular internalization of the CRISPR/Cas13a system and activation of gene editing upon entry into tumor tissue. After carefully screening and optimizing the CRISPR RNA (crRNA) sequence that targets programmed death-ligand 1 (PD-L1), DLNP demonstrates the effective activation of T-cell-mediated antitumor immunity and the reshaping of immunosuppressive tumor microenvironment (TME) in B16F10-bearing mice, resulting in significantly enhanced antitumor effect and improved survival rate. Further development by replacing the specific crRNA of target genes can potentially make DLNP a universal platform for the rapid development of safe and efficient cancer immunotherapies.

EGFR amplification and mutations are the most common oncogenic events in GBM. EGFR overexpression correlates with GBM invasion, but the underlying mechanisms are poorly understood. In a previous study, we showed that AJAP1 is involved in regulating F-actin to inhibit the invasive ability of GBM. In addition, in a GBM cell line, the AJAP1 promoter was highly bound by H3K27me3 and, through bioinformatics analysis, we found that AJAP1 expression was negatively correlated with EGFR. In this study, we found that the pathway downstream of EGFR had a higher activation level in GBM cell lines, which led to excessive tumor suppressor silencing. Therefore, we deduced that in glioma cells, the pathway downstream of EGFR remodels the cytoskeleton via AJAP1 epigenetic silencing to enhance invasion. Furthermore, MK2206 reversed AJAP1 downregulation by inhibiting the EGFR pathway. In vivo, MK2206 also inhibited the proliferation and local invasion of 87-EGFRvIII. These data suggest that activation of the EGFR signal transduction pathway genetically silences anti-oncogenes to enhance GBM malignancy. MK2206 might be a promising therapeutic for EGFR/EGFRvIII-positive GBMs.

RNA is rarely used as a therapeutic target due to its flexible structure and instability. CRISPR‐Cas13a is a powerful tool for RNA knockdown, and the potential application of CRISPR‐Cas13a in cancer cells should be further studied. In this study, overexpression of LwCas13a by lentivirus in glioma cells reveals that crRNA‐EGFP induces a “collateral effect” after knocking down the target gene in EGFP‐expressing cells. EGFRvIII is a unique EGFR mutant subtype in glioma, and the CRISPR‐Cas13a system induces death in EGFRvIII‐overexpressing glioma cells. Bulk and single‐cell RNA sequencing analysis in U87‐Cas13a‐EGFRvIII cells confirm the collateral effect of the CRISPR‐Cas13a system. Furthermore, CRISPR‐Cas13a inhibits the formation of glioma intracranial tumors in mice. The results demonstrate the collateral effect of the CRISPR‐Cas13a system in cancer cells and the powerful tumor‐eliminating potential of this system., It is reported that a CRISPR/Cas13a system can exert a collateral effect after target recognition in glioma cells, indicating that the CRISPR/Cas13a system has great potential value for tumor elimination, especially for patients with specific oncogene mutations.