CircRHBDD1 promotes immune escape via IGF2BP2/PD-L1 signaling and acts as a nanotherapeutic target in gastric cancer.

Circular RNAs (circRNAs) have been implicated in the development and progression of gastric cancer (GC). However, it remains unclear whether dysregulated circRNA affects immune escape and the efficacy of immunotherapy in GC. Our aim is to investigate the molecular mechanism of circRNA affecting GC immunotherapy and identify effective molecular therapeutic targets. The differential expression profile of circRNAs was established through circRNA sequencing, comparing three paired GC tissues with their adjacent non-cancerous gastric tissues. The expression level of circRHBDD1 in GC tissues was then assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The biological characteristics of circRHBDD1 were verified through a series of experiments, including agarose gel electrophoresis assays, RNase R treatment, and actinomycin D experiments. The prognostic value of circRHBDD1 in GC was evaluated by conducting both univariate and multivariate survival analyses. Furthermore, loss- and gain-of-function approaches were utilized to investigate the impact of circRHBDD1 on GC immune escape. RNA-sequencing, immunoprecipitation, flow cytometry, and methylated RNA immunoprecipitation (meRIP) analysis were performed to elucidate the underlying molecular mechanisms. We discovered that circRHBDD1 exhibited remarkably high expression levels in GC tissues and cell lines. Notably, the high expression of circRHBDD1 was significantly correlated with poor overall survival and disease-free survival among GC patients. Both in vitro and in vivo experiments revealed that circRHBDD1 upregulated the expression of PD-L1 and impeded the infiltration of CD8+ T cells. Further, we found that circRHBDD1 binds to IGF2BP2, disrupting the interaction between E3 ligase TRIM25 and IGF2BP2, and ultimately inhibiting IGF2BP2 ubiquitination and degradation. Intriguingly, IGF2BP2 enhances PD-L1 mRNA stability through m6A modification. Additionally, we developed Poly (lactide-co-glycolic acid) (PLGA)-Polyethylene glycol (PEG)-based nanoparticles loaded with circRHBDD1 siRNA. In vivo experiments validated that the combination of PLGA-PEG(si-circRHBDD1) and anti-PD-1 offers a safe and efficacious nano-drug regimen for cancer immunotherapy. Our results demonstrated that circRHBDD1 promoted GC immune escape by upregulating the expression of PD-L1 and reprogramming T cell-mediated immune response. Inhibition of circRHBDD1 expression could potentially enhance the response of GC patients to immunotherapy, thus improving treatment outcomes. Additionally, the development of a nanodrug delivery system provides a feasible approach for future clinical applications.

Li Y ，Wang Z ，Gao P ，Cao D ，Dong R ，Zhu M ，Fei Y ，Zuo X ，Cai J ... -  《Journal of Translational Medicine》

N(6)-methyladenosine-modified circIGF2BP3 inhibits CD8(+) T-cell responses to facilitate tumor immune evasion by promoting the deubiquitination of PD-L1 in non-small cell lung cancer.

An in-depth understanding of immune evasion mechanisms in tumors is crucial to overcome resistance and enable innovative advances in immunotherapy. Circular RNAs (circRNAs) have been implicated in cancer progression. However, much remains unknown regarding whether circRNAs impact immune escape in non-small-cell lung carcinoma (NSCLC). We performed bioinformatics analysis to profile and identify the circRNAs mediating immune evasion in NSCLC. A luciferase reporter assay, RNA immunoprecipitation (RIP), RNA pulldown assays and fluorescence in situ hybridization were performed to identify the interactions among circIGF2BP3, miR-328-3p, miR-3173-5p and plakophilin 3 (PKP3). In vitro T cell-mediated killing assays and in vivo syngeneic mouse models were used to investigate the functional roles of circIGF2BP3 and its downstream target PKP3 in antitumor immunity in NSCLC. The molecular mechanism of PKP3-induced PD-L1 upregulation was explored by immunoprecipitation, RIP, and ubiquitination assays. We demonstrated that circIGF2BP3 (hsa_circ_0079587) expression was increased in NSCLC and negatively correlated with CD8+ T cell infiltration. Functionally, elevated circIGF2BP3 inactivated cocultured T cells in vitro and compromised antitumor immunity in an immunocompetent mouse model, and this effect was dependent on CD8+ T cells. Mechanistically, METTL3 mediates the N6-methyladenosine (m6A) modification of circIGF2BP3 and promotes its circularization in a manner dependent on the m6A reader protein YTHDC1. circIGF2BP3 competitively upregulates PKP3 expression by sponging miR-328-3p and miR-3173-5p to compromise the cancer immune response. Furthermore, PKP3 engages with the RNA-binding protein FXR1 to stabilize OTUB1 mRNA, and OTUB1 elevates PD-L1 abundance by facilitating its deubiquitination. Tumor PD-L1 deletion completely blocked the impact of the circIGF2BP3/PKP3 axis on the CD8+ T cell response. The inhibition of circIGF2BP3/PKP3 enhanced the treatment efficacy of anti-PD-1 therapy in a Lewis lung carcinoma mouse model. Collectively, the PKP3/PD-L1 signature and the infiltrating CD8+ T cell status stratified NSCLC patients into different risk groups. Our results reveal the function of circIGF2BP3 in causing immune escape from CD8+ T cell-mediated killing through a decrease in PD-L1 ubiquitination and subsequent proteasomal degradation by stabilizing OTUB1 mRNA in a PKP3-dependent manner. This work sheds light on a novel mechanism of PD-L1 regulation in NSCLC and provides a rationale to enhance the efficacy of anti-PD-1 treatment in NSCLC.

Liu Z ，Wang T ，She Y ，Wu K ，Gu S ，Li L ，Dong C ，Chen C ，Zhou Y ... -  《Molecular Cancer》

N(6)-Methyladenosine (m(6)A) Reader IGF2BP1 Accelerates Gastric Cancer Development and Immune Escape by Targeting PD-L1.

N6-methyladenosine (m6A) functions as an important regulator in various human cancers, including gastric cancer. The immunotherapy targeting PD-1/PD-L1 has brought hope for advanced gastric cancer therapeutic. Here, present research aims to investigate the roles of m6A reader IGF2BP1 on gastric cancer tumor development and immune escape. Results indicated that IGF2BP1 up-regulated in the gastric cancer tissue and correlated with poor prognosis of gastric cancer patients. IGF2BP1 overexpression augmented the proliferation of co-cultured gastric cancer cells, and mitigated the CD8+ T cells mediated anti-tumor response, including IFN-γ secretion, surface PD-L1 level, and cytotoxicity of CD8+ T cells. Meanwhile, IGF2BP1 silencing exerted the opposite effects. In silico analysis revealed that there was a remarkable m6A modified site on PD-L1 mRNA. Moreover, the IGF2BP1 overexpression enhanced the stability of PD-L1 mRNA, thereby deteriorating the immune escape of gastric cancer cells. Collectively, these results describe a novel regulatory mechanism of IGF2BP1 by regulating PD-L1 through m6A epigenetic modification, which might provide insights for gastric cancer immunotherapies.

Tang B ，Bi L ，Xu Y ，Cao L ，Li X ... -  《-》

Overexpression of DAPK1-mediated inhibition of IKKβ/CSN5/PD-L1 axis enhances natural killer cell killing ability and inhibits tumor immune evasion in gastric cancer.

Gastric cancer (GC) originates from the stomach and is a prevalent human malignancy. Dysfunction of death associated protein kinase 1 (DAPK1) has been identified as a major regulator involved in the development and progression of GC. However, there's limited data regarding the regulatory mechanism of GC. Herein, we investigated role of DAPK1 in natural killer (NK) cell killing ability and immune evasion of GC cells and mediated pathway. Samples from GC-related gene expression profile and clinical samples from 67 patients with GC were collected to determine the expression of DAPK1, IκB kinase β (IKKβ), programmed death receptor-ligand 1 (PD-L1), and photomorphogenesis 9 (COP9) signalosome 5 (CSN5). The binding affinity among DAPK1, IKKβ, CSN5, and PD-L1 was characterized to verify the underlying mechanism. GC lines were transfected with overexpressed plasmid or siRNA to determine the effect of DAPK1/IKKβ/CSN5/PD-L1 axis on NK cell killing ability and immune evasion of GC cells. GC cells and tissues presented low expression of DAPK1 and high expression of IKKβ, CSN5 and PD-L1. IKKβ, negatively regulated by DAPK1, was capable of activating CSN5 and upregulating PD-L1 expression. Overexpression of DAPK1 promoted NK cell killing ability and reduced immune evasion, coupled with reduction of NK cell apoptosis and increases in levels of TNF-α, IFN-γ, CD107a, and Granzyme B cytokines. The tumor-suppressing properties of DAPK1 through downregulation of IKKβ/CSN5/PD-L1 axis in GC were further confirmed in vivo. In summary, overexpression of DAPK1 promoted the NK cell killing ability and restrained immune evasion of GC cells, providing a potential therapeutic strategy for GC treatment by modulating immune evasion.

Guo Z ，Zhou C ，Zhou L ，Wang Z ，Zhu X ，Mu X ... -  《-》

Circular RNA hsa_circ_0067842 facilitates tumor metastasis and immune escape in breast cancer through HuR/CMTM6/PD-L1 axis.

Circular RNAs (circRNAs) have been shown to play diverse biological functions in the progression of multiple diseases. However, the impacts of circRNAs on breast cancer (BC) progression remains unclear. Therefore, the objective of this paper is to investigate the role and mechanisms of a functional circRNA in BC metastasis and immune escape. This study used a circRNA microarray and identified a novel circRNA hsa_circ_0067842. The validation and characteristics of hsa_circ_0067842 were investigated using qRT-PCR, sanger sequencing, RNase R treatment, actinomycin D treatment and fluorescence in situ hybridization (FISH). Gain- and loss-of-function assays were performed to evaluate the biological function of hsa_circ_0067842 in BC progression and immune escape. Mechanistically, the interaction between hsa_circ_0067842 and HuR was explored by RNA pull down, mass spectrometry (MS), subcellular component protein extraction and immunofluorescence (IF). The regulatory mechanisms of hsa_circ_0067842/HuR/CMTM6/PD-L1 axis were investigated by qRT-PCR, western blot, FISH, immunoprecipitation and rescue assays. The expression of hsa_circ_0067842 was upregulated in BC tissues and cells, which was found to be significantly associated with poor prognosis, regardless of other clinical covariates. Function assays showed that hsa_circ_0067842 promoted the migration and invasion capacities of BC cells. Moreover, co-culture experiment with peripheral blood mononuclear cells (PBMCs) showed that hsa_circ_0067842 played a role in the immune escape of BC cells. Mechanistically, our study showed that hsa_circ_0067842 interacted with HuR, affecting its nuclear translocation, thus enhancing the stability of CMTM6. CMTM6 not only enhances the migration and invasion ability of BC cells, but also affects the ubiquitination of PD-L1 and inhibits its degradation. Collectively, our results demonstrated that hsa_circ_0067842 promoted BC progression through the HuR/CMTM6/PD-L1 axis, providing new insight and a potential target for BC prognosis and therapy.

Li J ，Dong X ，Kong X ，Wang Y ，Li Y ，Tong Y ，Zhao W ，Duan W ，Li P ，Wang Y ，Wang C ... -  《Biology Direct》