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Antisense oligonucleotide targeting TARDBP-EGFR splicing axis inhibits progression of oral squamous cell carcinoma
Nan Ni1,2, Moxu Wang1,2, Zhiran Yuan1,3
1Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, China.
Abstract:
Splice quantitative trait loci (sQTL) serve as another critical link between genetic variations and human diseases, besides expression quantitative trait loci (eQTL). Their role in oral squamous cell carcinoma (OSCC) development remains unexplored. We collected surgically resected cancer and adjacent normal epithelial tissue samples from 67 OSCC cases, and extracted RNA for sequencing after quality control. A genome-wide sQTL analysis was performed using the RNA sequencing data from 67 normal oral epithelial tissue samples. We included peripheral blood DNA samples from 1044 patients with OSCC and 3199 healthy controls to conduct a genome-wide association study. Systematic screening of sQTLs associated with OSCC risk identified a sQTL variant-the rs737540-T allele-independent of eQTLs, significantly associated with an increased risk of OSCC (OR = 1.2, P = 6.84 × 10-4). The rs737540-T allele reduced skipping of EGFR alternative exon 4 by enhancing TAR DNA binding protein (TARDBP) binding to the RNA sequence, leading to increased expression of the longer isoform (EGFR-001) and reduced expression of the truncated isoform (EGFR-004). Compared with EGFR-004, EGFR-001 promoted OSCC cell proliferation by reducing ATP-binding cassette subfamily A member 1 (ABCA1) ubiquitination through lower EGFR phosphorylation. ABCA1 was demonstrated to increase the cholesterol content of the plasma membrane via cholesterol efflux, thus affecting membrane fluidity and vimentin-mediated epithelial-mesenchymal transition. An antisense oligonucleotide targeting rs737540 significantly inhibited OSCC proliferation and reversed membrane cholesterol-induced resistance. This study provides novel insights into how genetic variants regulating alternative splicing contribute to OSCC risk and identifies potential therapeutic targets.
Insights
Genetic variants influencing RNA splicing, or splice quantitative trait loci (sQTLs), are linked to oral squamous cell carcinoma (OSCC) risk. A specific sQTL variant, rs737540-T, increases OSCC risk by altering EGFR splicing and promoting cancer cell growth.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Splice quantitative trait loci (sQTLs) are crucial genetic factors linking variations to diseases, similar to expression quantitative trait loci (eQTLs).
- The role of sQTLs in the development of oral squamous cell carcinoma (OSCC) has not been previously investigated.
Purpose of the Study:
- To explore the association between sQTLs and the risk of developing oral squamous cell carcinoma (OSCC).
- To elucidate the molecular mechanisms by which identified sQTLs influence OSCC progression.
- To identify potential therapeutic targets for OSCC based on sQTL-driven pathways.
Main Methods:
- Genome-wide sQTL analysis using RNA sequencing data from 67 normal oral epithelial tissues.
- Genome-wide association study (GWAS) using DNA samples from 1044 OSCC patients and 3199 healthy controls.
- Functional assays to investigate the impact of the sQTL variant on gene splicing, protein expression, and cellular processes.
Main Results:
- A novel sQTL variant, rs737540-T, was identified as significantly associated with increased OSCC risk (OR=1.2, P=6.84×10-4), independent of eQTLs.
- The rs737540-T allele enhances TAR DNA binding protein (TARDBP) binding, reducing EGFR alternative exon 4 skipping and increasing the expression of the full-length EGFR isoform (EGFR-001).
- EGFR-001 promotes OSCC proliferation by altering ABCA1 ubiquitination and cholesterol metabolism, impacting membrane fluidity and epithelial-mesenchymal transition; an antisense oligonucleotide targeting rs737540 inhibited OSCC proliferation.
Conclusions:
- Genetic variants regulating alternative splicing, specifically rs737540, contribute significantly to OSCC risk.
- The rs737540-mediated alteration in EGFR splicing and downstream effects on cholesterol metabolism represent a novel mechanism in OSCC development.
- Targeting rs737540 offers a potential therapeutic strategy for inhibiting OSCC proliferation.
