BackgroundThe therapeutic application of T cells endowing with chimeric antigen receptors (CARs) is faced with “on-target, off-tumor” toxicity against solid tumors, particularly in the treatment of the pancreatic cancer. To our best knowledge, the pancreatic cancer cell line AsPC-1 often highly expressed some distinct tumor-associated antigens, such as carcino-embryonic antigen (CEA) and mesothelin (MSLN). Therefore, in this research, we have characterized dual-receptor CAR-modified T cells (dCAR-T) that exert effective and safe cytotoxicity against AsPC-1 cells.MethodsBased on the dual signaling pathway of wild T cells, we designed a novel dCAR diagram specific for CEA and MSLN, which achieved comparable activity relative to that of conventional CAR-T cells (CEA-CAR T or MSLN-CAR T). In this dCAR, a tandem construct containing two physically separate structures, CEA-CD3ζ and MSLN-4/1BB signaling domains were effectively controlled with tumor antigens CEA and MSLN, respectively. Finally, the activity of dCAR-T cells has been verified via in vitro and in vivo experiments.ResultsIn the presence of cognate tumor cells (AsPC-1) expressing both CEA and MSLN, dCAR-T cells exerted high anti-tumor activity relative to that of other single-receptor CAR-T cells bearing only one signaling pathway (e.g., Cζ-CAR and MBB-CAR). In a xenograft model, dCAR-T cells significantly inhibited the growth of AsPC-1 cells yet no effect on the growth of non-cognate tumor cells. Furthermore, the released cytokines and T cell persistence in mice were comparable with that of conventional CAR-T cells, obtaining specific and controllable cytotoxicity.ConclusionsA novel type of CAR-T cells, termed dCAR-T, was designed with specific activities, that is, significant cytotoxicity for two antigen-positive tumor cells yet no cytotoxicity for single antigen-positive tumor cells. Dual-targeted CAR-T cells can be precisely localized at the tumor site and can exert high cytotoxicity against tumor cells, alleviating “on-target, off-tumor” toxicity and enabling accurate application of CAR-T cell therapy.Electronic supplementary materialThe online version of this article (10.1186/s13045-018-0646-9) contains supplementary material, which is available to authorized users.

Background aims Chimeric antigen receptor T cells (CAR‐T cells) have been successfully used in treatments of hematological tumors, however, their anti‐tumor activity in solid tumor treatments was limited. As IL‐12 increases T‐cell immune functions, we designed carcinoembryonic antigen (CEA) specific CAR‐T (CEA‐CAR‐T) cells and, for the first time, used them in combination with recombinant human IL‐12 (rhIL‐12) to treat several types of solid tumors. Methods In vitro anti‐tumor activity of CEA‐CAR‐T cells in combination with rhIL‐12 was confirmed by evaluation of CEA‐CAR‐T cell activation, proliferation, and cytotoxicity after co‐incubation with CEA‐positive or CEA‐negative human tumor cells. In vivo anti‐tumor activity of CEA‐CAR‐T cells in combination with rhIL‐12 was confirmed in a xenograft model in nude mice for treatments of several types of solid tumors. Results In vitro experiments confirmed that rhIL‐12 significantly increased the activation, proliferation, and cytotoxicity of CEA‐CAR‐T cells. Similarly, in vivo experiments found that CEA‐CAR‐T cells in combination with rhIL‐12 had significantly enhanced anti‐tumor activity than CEA‐CAR‐T cells in growth inhibition of newly colonized colorectal cancer cell HT‐29, pancreatic cancer cell AsPC‐1, and gastric cancer cell MGC803. Conclusions These works confirmed that simultaneous use of cytokines, for example, rhIL‐12, can increase the anti‐tumor activity of CAR‐T cells, especially for treatments of several types of solid tumors.

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Natural killer (NK) cells have been suggested to be associated with type 2 diabetes by regulating systemic inflammation. However, the mechanism by which NK cells regulate insulin sensitivity remains unknown. This study shows that NK-derived exosomes from lean mice attenuate obesity-induced insulin resistance and inflammation in mice of type 2 diabetes. Moreover, lean NK-derived exosomes enhance insulin sensitivity and relieve inflammation in adipocytes and hepatocytes. MiR-1249-3p, which is significantly upregulated in lean NK-derived exosomes, can be transferred from NK cells to adipocytes and hepatocytes via exosomes. NK-derived exosomal miR-1249-3p dramatically induces cellular insulin sensitivity and relieves inflammation. Mechanistically, exosomal miR-1249-3p directly targets SKOR1 to regulate the formation of ternary complex SMAD6/MYD88/SMURF1, which mediates glucose homeostasis by suppressing the TLR4/NF-κB signaling pathway. This study reveals an emerging role for NK-derived exosomal miR-1249-3p in remission of insulin resistance, and provides a series of potential therapeutic targets in type 2 diabetes.

Long non-coding RNAs (lncRNAs) contribute to the initiation and progression of various tumors, including head and neck squamous carcinoma (HNSCC), which is a common malignancy with high morbidity and low survival rate. However, the mechanism of lncRNAs in HNSCC tumorigenesis remains largely unexplored. In this work, we identified a novel lncRNA AC104041.1 which is highly upregulated and correlated with poor survival in HNSCC patients. Moreover, AC104041.1 overexpression significantly promoted tumor growth and metastasis of HNSCC in vitro and in vivo. Mechanistically, AC104041.1 mainly located in the cytoplasm and could function as ceRNA (competing endogenous RNA) for miR-6817-3p, thereby stabilized Wnt2B, and consequently inducing β-catenin nuclear translocation and activation. Moreover, we demonstrate that salinomycin, which as a highly effective antibiotic in the elimination of cancer stem cells through the Wnt/β-catenin signaling, could enhance the inhibition of tumor growth by antisense oligonucleotides (ASO) targeting AC104041.1 in HNSCC cells and PDXs (patient-derived xenograft) model. Thus, our data provide preclinical evidence to support a novel strategy of ASOs targeting AC104041.1 in combination with salinomycin and may as a beneficial treatment approach for HNSCC.

Several important micropeptides encoded by noncoding RNAs have been identified in recent years; however, there have never been any reports of micropeptides in head and neck squamous cell carcinoma (HNSCC). Here we report the discovery and characterization of a human endogenous peptide named micropeptide inhibiting actin cytoskeleton (MIAC). Comprehensive analysis of the TCGA (The Cancer Genome Atlas) database (n = 500), clinical fresh samples (n = 94), and tissue microarrays (n = 60) revealed that lower MIAC expression is correlated with poor overall survival of HNSCC patients. Meanwhile, RNA-sequencing analysis of 9657 human tissues across 32 cancer types from TCGA cohorts found that MIAC is significantly associated with the progression of 5 other different tumors. Mechanistically, MIAC directly interacts with AQP2 (Aquaporin 2) to inhibit the actin cytoskeleton by regulating SEPT2 (Septin 2)/ITGB4 (Integrin Beta 4) and ultimately suppressing the tumor growth and metastasis of HNSCC. Collectively, the mechanism investigation and evaluation of MIAC activity in vivo and in vitro highlights that MIAC plays an important role in HNSCC tumorigenesis.

BackgroundApigenin, as a natural flavonoid, has low intrinsic toxicity and has potential pharmacological effects against hepatocellular carcinoma (HCC). However, the molecular mechanisms involving microRNAs (miRNAs) and their target genes regulated by apigenin in the treatment of HCC have not been addressed.ObjectiveIn this study, the molecular mechanisms of apigenin involved in the prevention and treatment of HCC were explored in vivo and in vitro using miRNA transcriptomic sequencing to determine the basis for the clinical applications of apigenin in the treatment of HCC.MethodsThe effects of apigenin on the proliferation, cell cycle progression, apoptosis, and invasion of human hepatoma cell line Huh7 and Hep3B were studied in vitro, and the effects on the tumorigenicity of Huh7 cells were assessed in vivo. Then, a differential expression analysis of miRNAs regulated by apigenin in Huh7 cells was performed using next-generation RNA sequencing and further validated by qRT-PCR. The potential genes targeted by the differentially expressed miRNAs were identified using a curated miRTarBase miRNA database and their molecular functions were predicted using Gene Ontology and KEGG signaling pathway analysis.ResultsCompared with the control treatment group, apigenin significantly inhibited Huh7 cell proliferation, cell cycle, colony formation, and cell invasion in a concentration-dependent manner. Moreover, apigenin reduced tumor growth, promoted tumor cell necrosis, reduced the expression of Ki67, and increased the expression of Bax and Bcl-2 in the xenograft tumors of Huh7 cells. Bioinformatics analysis of the miRNA transcriptome showed that hsa-miR-24, hsa-miR-6769b-3p, hsa-miR-6836-3p, hsa-miR-199a-3p, hsa-miR-663a, hsa-miR-4739, hsa-miR-6892-3p, hsa-miR-7107-5p, hsa-miR-1273g-3p, hsa-miR-1343, and hsa-miR-6089 were the most significantly up-regulated miRNAs, and their key gene targets were MAPK1, PIK3CD, HRAS, CCND1, CDKN1A, E2F2, etc. The core regulatory pathways of the up-regulated miRNAs were associated with the hepatocellular carcinoma pathway. The down-regulated miRNAs were hsa-miR-181a-5p and hsa-miR-148a-3p, and the key target genes were MAPK1, HRAS, STAT3, FOS, BCL2, SMAD2, PPP3CA, IFNG, MET, and VAV2, with the core regulatory pathways identified as proteoglycans in cancer pathway.ConclusionApigenin can inhibit the growth of HCC cells, which may be mediated by up-regulation or down-regulation of miRNA molecules and their related target genes.

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