Reduced YTHDF2 inhibits PD-L1 expression by stabilizing m⁶A-containing SPOP mRNA in colorectal cancer

Area of Science:

• Oncology
• Molecular Biology
• Immunology

Background:

• Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide.
• The regulatory mechanisms of programmed death ligand 1 (PD-L1) in CRC remain incompletely understood.
• YTH N⁶-methyladenosine (m⁶A) RNA binding protein F2 (YTHDF2) is implicated in various cancers, but its role in CRC progression and PD-L1 regulation is unclear.

Purpose of the Study:

• To investigate the function of YTHDF2 in colorectal cancer development and progression.
• To elucidate the molecular mechanism by which YTHDF2 influences PD-L1 expression in CRC.
• To explore the potential of the YTHDF2-SPOP-PD-L1 axis as a therapeutic target for CRC.

Main Methods:

• Analysis of gene expression profiling datasets and clinical CRC samples.
• In vivo studies using a subcutaneous immunodeficient mouse model of CRC.
• In vitro assays including Western blots, flow cytometry, PD-1/PD-L1 binding assays, and cell killing assays.
• RNA sequencing (RNA-seq), methylated RNA immunoprecipitation (MeRIP) sequencing, and RNA binding protein immunoprecipitation (RIP) sequencing to identify YTHDF2 targets and m⁶A modification sites.
• Bioinformatic analysis, SRAMP, MeRIP-qRT-PCR, RIP-qRT-PCR, and dual-luciferase reporter assays for validation.

Main Results:

• YTHDF2 expression was significantly upregulated in CRC tissues, correlating with a poorer patient prognosis.
• YTHDF2 promoted CRC tumor growth in vivo, but did not affect cell proliferation, migration, or invasion in vitro.
• Interference with YTHDF2 expression led to reduced PD-L1 levels and impaired PD-1/PD-L1 binding.
• Speckle type BTB/POZ protein (SPOP) mRNA was identified as a direct YTHDF2 target, with m⁶A methylation sites validated.
• YTHDF2 knockdown increased SPOP mRNA stability and expression, subsequently decreasing PD-L1 expression.

Conclusions:

• YTHDF2 promotes colorectal cancer progression by destabilizing SPOP mRNA, which leads to increased PD-L1 expression.
• The YTHDF2-SPOP-PD-L1 signaling axis represents a novel regulatory pathway in CRC.
• Targeting YTHDF2 or its downstream effectors could offer a promising therapeutic strategy for CRC and enhance anti-PD-1/PD-L1 immunotherapy efficacy.