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

## TL;DR

This study shows that YTHDF2 promotes colorectal cancer by increasing PD-L1 levels through destabilizing SPOP mRNA, offering a new target for cancer treatment.

## Contribution

The study reveals a novel YTHDF2-SPOP-PD-L1 regulatory axis in colorectal cancer.

## Key findings

- YTHDF2 is upregulated in CRC and linked to worse patient outcomes.
- Reduced YTHDF2 stabilizes SPOP mRNA, decreasing PD-L1 expression.
- The YTHDF2-SPOP-PD-L1 axis is a potential therapeutic target for CRC.

## Abstract

Colorectal cancer (CRC) is one of the most frequently diagnosed malignant tumors. However, clear evidence explaining the regulatory mechanisms of programmed death ligand 1 (PD-L1) in CRC has been limited. To illustrate the function of YTH N6-methyladenosine (m6A) RNA binding protein F2 (YTHDF2), we conducted a comprehensive evaluation of expression profiling datasets from online databases and clinical samples. We used a subcutaneous immunodeficient mouse model to investigate the impact of YTHDF2 on CRC. Western blots, flow cytometry, PD-1/PD-L1 binding assay, and cell killing assay were used to assess the relationship between YTHDF2 and PD-L1. We used RNA sequencing, along with methylated RNA immunoprecipitation (MeRIP) and RNA binding protein immunoprecipitation (RIP) sequencing to analyze mRNA expression, m6A methylation levels, and YTHDF2 target transcripts. The m6A methylation locations of mRNAs were verified using sequence-based RNA adenosine methylation site predictor (SRAMP), MeRIP-qRT-PCR, RIP-qRT-PCR, and a dual-luciferase reporter system. YTHDF2 was upregulated in CRC tissues, and patients with higher YTHDF2 expression had a worse prognosis. The in vivo model showed that YTHDF2 promoted CRC growth, whereas in vitro experiments showed that inhibiting YTHDF2 expression did not affect cell proliferation, migration, or invasion. Mechanistically, interference with YTHDF2 reduced PD-L1 expression and the binding ability between PD-1 and PD-L1. The use of RNA-seq, MeRIP-seq, RIP-seq, and bioinformatics tools confirmed that the speckle type BTB/POZ protein (SPOP) mRNA was a YTHDF2 target and validated its m6A methylation sites. After YTHDF2 knockdown, SPOP mRNA stability increased, causing an increase in SPOP expression and a decrease in PD-L1 expression. This study demonstrated that YTHDF2 might upregulate PD-L1 expression by destabilizing m6A-containing SPOP mRNA and promote CRC development. The biological effect of the YTHDF2-SPOP-PD-L1 axis presented a promising target for CRC treatment and provided an approach to enhance the efficacy of anti-PD-1/PD-L1 therapy.