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The application of mRNA as a novel kind of vaccine has been proved recently, due to the emergence use authorization (EUA) by FDA for the two COVID-19 mRNA vaccines developed by Moderna and BioNTech. Both of the two vaccines are based on canonical linear mRNA, and encapsulated by lipid nanoparticle (LNP). Circular mRNA, which is found to mediate potent and durable protein expression, is an emerging technology recently. Owing to its simplicity of manufacturing and superior performance of protein expression, circular mRNA is believed to be a disruptor for mRNA area. However, the application of circular mRNA is still at an initiation stage, proof of concept for its usage as future medicine or vaccine is necessary. In the current study, we established a novel kind of circular mRNA, termed C-RNA, based on Echovirus 29 (E29)-derived internal ribosome entry sites (IRES) and newly designed homology arms and RNA spacers. Our results demonstrated that this kind of circular mRNA is able to mediate strong and durable protein expression, compared to typical linear mRNA. Moreover, for the first time, our study demonstrated that direct intratumoral administration of C-RNA encoding a mixture of cytokines achieved successful modulation of intratumoral and systematic anti-tumor immune responses and finally leading to an enhancement of anti-PD-1 antibody-induced tumor repression in syngeneic mouse model. Additionally, after an optimization of the circular mRNA formulation, a significant improvement of C-RNA mediated protein expression was observed. With this optimized formulation, C-RNA induced enhanced anti-tumor effect via intratumoral administration and elicited significant activation of tumor-infiltrated total T cells and CD8+ T cells. Collectively, we established C-RNA, a novel circular mRNA platform, and demonstrated that it can be applied for direct intratumoral administration for cancer therapy.

Summary The mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cellular innate immunity, metabolism, and senescence. FK506-binding protein 12 (FKBP12) inhibits mTOR kinase activity via direct association. The FKBP12-mTOR association can be strengthened by the immunosuppressant rapamycin, but the underlying mechanism remains elusive. We show here that the FKBP12-mTOR association is tightly regulated by an acetylation–deacetylation cycle. FKBP12 is acetylated on the lysine cluster (K45/K48/K53) by CREB-binding protein (CBP) in mammalian cells in response to nutrient treatment. Acetyl-FKBP12 associates with CBP acetylated Rheb. Rapamycin recruits SIRT2 with a high affinity for FKBP12 association and deacetylation. SIRT2-deacetylated FKBP12 then switches its association from Rheb to mTOR. Nutrient-activated mTOR phosphorylates IRF3S386 for the antiviral response. In contrast, rapamycin strengthening FKBP12-mTOR association blocks mTOR antiviral activity by recruiting SIRT2 to deacetylate FKBP12. Hence, on/off mTOR activity in response to environmental nutrients relies on FKBP12 acetylation and deacetylation status in mammalian cells., Graphical abstract, Highlights • FKBP12-mTOR association is tightly regulated by an acetylation–deacetylation cycle • SIRT2 is responsible for FKBP12 deacetylation • Acetylation of Rheb is indispensable to mTOR activation • mTOR phosphorylates IRF3 S386 for type-I interferon gene expression, Biochemistry; Protein; Molecular biology

Background LOX-like 1 (LOXL1) is a lysyl oxidase, and emerging evidence has revealed its effect on malignant cancer progression. However, its role in colorectal cancer (CRC) and the underlying molecular mechanisms have not yet been elucidated. Methods LOXL1 expression in colorectal cancer was detected by immunohistochemistry, western blotting and real-time PCR. In vitro, colony formation, wound healing, migration and invasion assays were performed to investigate the effects of LOXL1 on cell proliferation, migration and invasion. In vivo, metastasis models and mouse xenografts were used to assess tumorigenicity and metastasis ability. Molecular biology experiments were utilized to reveal the underlying mechanisms by which LOXL1 modulates the Hippo pathway. Results LOXL1 was highly expressed in normal colon tissues compared with cancer tissues. In vitro, silencing LOXL1 in CRC cell lines dramatically enhanced migration, invasion, and colony formation, while overexpression of LOXL1 exerted the opposite effects. The results of the in vivo experiments demonstrated that the overexpression of LOXL1 in CRC cell lines drastically inhibited metastatic progression and tumour growth. Mechanistically, LOXL1 inhibited the transcriptional activity of Yes-associated protein (YAP) by interacting with MST1/2 and increasing the phosphorylation of MST1/2. Conclusions LOXL1 may function as an important tumour suppressor in regulating tumour growth, invasion and metastasis via negative regulation of YAP activity. Graphical abstract

Additional file 5: Figure S5. YAP can reverse the inhibitory effect of LOXL1. Transwell migration and Matrigel invasion assays were performed in LOXL1 alone or LOXL1 and YAP co-transfected HCT8 cells. Representative images (left panel) and quantification (right panel) are shown as indicated. Data from three independent experiments are presented as the mean ± SD. Statistical significance was assessed by unpaired t-test; **P

Additional file 2: Figure S2. Knockdown of LOXL1 in RKO and HT29 cells increases their proliferation ability in vitro. CCK8 analysis was performed to detect the cell proliferation. Data from three independent experiments are presented as the mean ± SD. Statistical significance was assessed by unpaired t-test; ***P

Additional file 1: Figure S1. LOXL1 overexpression inhibits the proliferation of CRC cells. a HCT8-N/HCT8-LOXL1 and SW480-N/SW480-LOXL1 cells were detected by CCK8 analysis. Data are shown as the mean ± SD of triplicate independent sets of experiments; statistical significance was assessed by unpaired t-test. ***P

Additional file 3: Figure S3. LOXL1 negatively regulates the YAP activity. a LOXL1 and YAP constructs alone or combined, as indicated, were transfected into HEK293T cells together with a indicated luciferase reporters. The results shown were normalized for transfection efficiency. Data are shown as the mean ± SD of triplicate independent sets of experiments; statistical significance was assessed by unpaired t-test. ns; non-significant, ***P

Additional file 6: Figure S6. a H-E staining of HCT8-N/HCT8-LOXL1 and SW480-N/SW480-LOXL1 xenograft tumours. b and c HCT8-N/HCT8-LOXL1 and SW480-N/SW480-LOXL1 xenograft tumours were detected the p-YAP(S127) and Ki67 levels by immunohistochemistry. d Cell apoptosis in xenografted tumors was measured by the TUNEL assay. Scale bar: 100 μm.

Additional file 4: Figure S4 Knockdown of LOXL1 decreases the Hippo pathway activation. HT29 cells were transfected with siRNA to LOXL1 or a control siRNA (N) for 48 h. Cell lysates were analysed by immunoblotting with the indicated antibodies.