N6-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

Abstract

Background 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). Methods 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. Results 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. Conclusion 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.

m6A Modification in Non-Coding RNA: The Role in Cancer Drug Resistance

“…m6A-modified circIGF2BP3 inhibited the activity of T cells in vitro and restrain antitumor immunity in vivo by upregulating PKP3 to elevate PD-L1 abundance. Furthermore, the inhibition of circIGF2BP3/PKP3 enhanced the effects of anti-PD-1 therapy in a Lewis lung carcinoma mouse model ( 158 ). These works suggested that m6A regulators or m6A-modified non-coding RNA could be a therapeutic target for immunotherapy in combination with PD-L1 inhibitors.…”

CircRNA-DOPEY2 enhances the chemosensitivity of esophageal cancer cells by inhibiting CPEB4-mediated Mcl-1 translation

“…Nuclear and cytoplasmic extraction reagents (NE-PER, Pierce, IL, USA) were employed to isolate fractional RNA in the cytoplasm and nucleus. Real-time qPCR was performed using SYBR Premix Ex Taq (TaKaRa Bio, Beijing, China) as previously described [ 16 ]. The expression levels of the RNAs of interest were normalized to those of GAPDH, and the primers used in this study are listed in Table S 3 .…”

The Role of Circular RNAs in the Drug Resistance of Cancers

“…CircIGF2BP3 expression is negatively associated with the infiltration of CD8 + T cells, which induces immune escape from CD8 + T cell-mediated killing. Mechanistically, PKP3 upregulated by circIGF2BP3 combines with FXR1 to stabilize OTUB1 mRNA, which increases PD-L1 abundance by promoting its deubiquitination ( 39 ). A study on ovarian cancer has suggested that silencing of circ_C20orf11 suppresses extracellular vesicle (EV)-induced macrophage M2 polarization and enhances sensitivity to CDDP in vivo ( 40 ).…”