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Role of PCIF1-mediated 5′-cap N6-methyladeonsine mRNA methylation in colorectal cancer and anti-PD-1 immunotherapy

ABSTRACT: Adenosine N6-methylation (m6A) and N6,2′-O-dimethylation (m6Am) are regulatory modifications of eukaryotic mRNAs. m6Am formation is catalysed by the methyl transferase phosphorylated CTD-interacting factor 1 (PCIF1); however, the pathophysiological functions of this RNA modification and PCIF1 in cancers are unclear. Here, we show that PCIF1 expression is upregulated in colorectal cancer (CRC) and negatively correlates with patient survival. CRISPR/Cas9-mediated depletion of PCIF1 in human CRC cells leads to loss of cell migration, invasion, and colony formation in vitro and loss of tumor growth in athymic mice. Pcif1 knockout in murine CRC cells inhibits tumor growth in immunocompetent mice and enhances the effects of anti-PD-1 antibody treatment by decreasing intra-tumoral TGF-β levels, and increasing intra-tumoral IFN-γ, TNF-α levels and tumor-infiltrating natural killer cells. We further show that PCIF1 modulates CRC growth and response to anti-PD-1 in a context dependent mechanism with PCIF directly targeting FOS, IFITM3, and STAT1 via m6 Am modifications. PCIF1stabilizes FOS mRNA, which in turn leads to FOS-dependent TGF-β regulation and tumor growth. While during immunotherapy, Pcif1-Fos-TGF-β, as well as Pcif1-Stat1/Ifitm3-IFN-γ axes contribute to the resistance of anti-PD-1 therapy. Collectively, our findings reveal a role of PCIF1 in promoting CRC tumorigenesis and resistance to anti-PD-1 therapy, supporting that the combination of PCIF1 inhibition with anti-PD-1 treatment is a potential therapeutic strategy to enhancement CRC response to immunotherapy. Finally, we developed a lipid-nanoparticles (LNPs) and chemically modified small interfering RNAs (CMsiRNAs)-based strategy to silence PCIF1 in vivo and found that this treatment significantly reduced tumor growth in mice. Our results therefore provide a proof-of-concept for tumor growth suppression using LNP-CMsiRNA to silence target genes in cancer.

SUBMITTER: Dr. Lingling Wang

PROVIDER: S-SCDT-10_15252-EMBJ_2022111673 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:BackgroundColorectal cancer (CRC) is one of the leading causes of tumor-related death worldwide, and its main cause of death is distant metastasis. Methyltransferase-like 14(METTL14), a major RNA N6-adenosine methyltransferase, is involved in tumor progression via regulating RNA function. The goal of the study is to uncover the biological function and molecular mechanism of METTL14 in CRC.MethodsQuantitative real-time PCR (qRT-PCR), western blot and immunohistochemical (IHC) assays were employed to detect METTL14 and SOX4 in CRC cell lines and tissues. The biological functions of METTL14 were demonstrated using in vitro and in vivo experiments. Chromatin immunoprecipitation (ChIP), Transcrptomic RNA sequencing (RNA-Seq), m6A-RNA immunoprecipitation sequencing (MeRIP-Seq), RNA immunoprecipitation and luciferase reporter assays were used to explore the mechanism of METTL14 action.ResultsMETTL14 expression was significantly downregulated in CRC and decreased METTL14 was associated with poor overall survival (OS). Both the univariate and multivariate Cox regression analysis indicated that METTL14 was an independent prognostic factor in CRC. Moreover, lysine-specific histone demethylase 5C(KDM5C)-mediated demethylation of histone H3 lysine 4 tri-methylation(H3K4me3) in the promoter of METTL14 inhibited METTL14 transcription. Functionally, we verified that METTL14 inhibited CRC cells migration, invasion and metastasis through in vitro and in vivo assays, respectively. Furthermore, we identified SRY-related high-mobility-group box 4(SOX4) as a target of METTL14-mediated m6A modification. Knockdown of METTL14 markedly abolished SOX4 mRNA m6A modification and elevated SOX4 mRNA expression. We also revealed that METTL14-mediated SOX4 mRNA degradation relied on the YTHDF2-dependent pathway. Lastly, we demonstrated that METTL14 might inhibit CRC malignant process partly through SOX4-mediated EMT process and PI3K/Akt signals.ConclusionsDecreased METTL14 facilitates tumor metastasis in CRC, suggesting that METTL14 might be a potential prognostic biomarker and effective therapeutic target for CRC.

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Role of PCIF1-mediated 5′-cap N6-methyladeonsine mRNA methylation in colorectal cancer and anti-PD-1 immunotherapy

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