Antisense oligonucleotide targeting TARDBP-EGFR splicing axis inhibits progression of oral squamous cell carcinoma through ABCA1-regulated cholesterol efflux

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.

References

[1] Tan Y, et al.. Oral squamous cell carcinomas: state of the field and emerging directions. Int J. Oral. Sci., 2023, 15: 44

[2] Abou Faycal C, Gazzeri S, Eymin B. RNA splicing, cell signaling, and response to therapies. Curr. Opin. Oncol., 2016, 28: 58-64

[3] Wright CJ, Smith CWJ, Jiggins CD. Alternative splicing as a source of phenotypic diversity. Nat. Rev. Genet., 2022, 23: 697-710

[4] Kahles A, et al.. Comprehensive analysis of alternative splicing across tumors from 8,705 patients. Cancer Cell, 2018, 34: 211-224.e216

[5] Bradley RK, Anczukow O. RNA splicing dysregulation and the hallmarks of cancer. Nat. Rev. Cancer, 2023, 23: 135-155

[6] Consortium GT. The GTEx Consortium atlas of genetic regulatory effects across human tissues. Science, 2020, 369: 1318-1330

[7] Zhang M, et al.. Genetic control of alternative splicing and its distinct role in colorectal cancer mechanisms. Gastroenterology, 2023, 165: 1151-1167

[8] Wang Y, et al.. Integrative splicing-quantitative-trait-locus analysis reveals risk loci for non-small-cell lung cancer. Am. J. Hum. Genet, 2023, 110: 1574-1589

[9] Byeon HK, Ku M, Yang J. Beyond EGFR inhibition: multilateral combat strategies to stop the progression of head and neck cancer. Exp. Mol. Med., 2019, 51: 1-14

[10] Ciardiello F, Tortora G. EGFR antagonists in cancer treatment. N. Engl. J. Med., 2008, 358: 1160-1174

[11] Johnson DE, et al.. Head and neck squamous cell carcinoma. Nat. Rev. Dis. Prim., 2020, 6: 92

[12] Rampias T, et al.. RAS/PI3K crosstalk and cetuximab resistance in head and neck squamous cell carcinoma. Clin. Cancer Res., 2014, 20: 2933-2946

[13] Maemondo M, et al.. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N. Engl. J. Med., 2010, 362: 2380-2388

[14] Paik PK, et al.. Tepotinib in non-small-cell lung cancer with MET Exon 14 skipping mutations. N. Engl. J. Med, 2020, 383: 931-943

[15] Nomakuchi TT, Rigo F, Aznarez I, Krainer AR. Antisense oligonucleotide-directed inhibition of nonsense-mediated mRNA decay. Nat. Biotechnol., 2016, 34: 164-166

[16] Dewaele M, et al.. Antisense oligonucleotide-mediated MDM4 exon 6 skipping impairs tumor growth. J. Clin. Invest, 2016, 126: 68-84

[17] Dong YS, et al.. Unexpected requirement for a binding partner of the syntaxin family in phagocytosis by murine testicular Sertoli cells. Cell Death Differ., 2016, 23: 787-800

[18] Lei K, et al.. Cancer-cell stiffening via cholesterol depletion enhances adoptive T-cell immunotherapy. Nat. Biomed. Eng., 2021, 5: 1411-1425

[19] Consortium GT, et al.. Genetic effects on gene expression across human tissues. Nature, 2017, 550: 204-213

[20] Morley M, et al.. Genetic analysis of genome-wide variation in human gene expression. Nature, 2004, 430: 743-747

[21] Yao DW, O’Connor LJ, Price AL, Gusev A. Quantifying genetic effects on disease mediated by assayed gene expression levels. Nat. Genet, 2020, 52: 626-633

[22] Pavlides JM, et al.. Predicting gene targets from integrative analyses of summary data from GWAS and eQTL studies for 28 human complex traits. Genome Med., 2016, 8 84

[23] Walker RL, et al.. Genetic control of expression and splicing in developing human brain informs disease mechanisms. Cell, 2020, 181: 745

[24] Li YI, et al.. RNA splicing is a primary link between genetic variation and disease. Science, 2016, 352: 600-604

[25] Xiao M, et al.. Functional significance of cholesterol metabolism in cancer: from threat to treatment. Exp. Mol. Med, 2023, 55: 1982-1995

[26] Yue S, et al.. Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness. Cell Metab., 2014, 19: 393-406

[27] Du L, et al.. Metabolomic profiling of plasma reveals potential biomarkers for screening and early diagnosis of gastric cancer and precancerous stages. MedComm – Oncol., 2023, 2: e32

[28] Du A, et al.. Fatty acids in cancer: Metabolic functions and potential treatment. MedComm – Oncol., 2023, 2: e25

[29] Pan Z, et al.. Cholesterol promotes EGFR-TKIs resistance in NSCLC by inducing EGFR/Src/Erk/SP1 signaling-mediated ERRalpha re-expression. Mol. Cancer, 2022, 21 77

[30] Kuzu OF, Noory MA, Robertson GP. The role of cholesterol in cancer. Cancer Res, 2016, 76: 2063-2070

[31] Liu X, Lv M, Zhang W, Zhan Q. Dysregulation of cholesterol metabolism in cancer progression. Oncogene, 2023, 42: 3289-3302

[32] Lindow M, et al.. Assessing unintended hybridization-induced biological effects of oligonucleotides. Nat. Biotechnol., 2012, 30: 920-923

[33] Kamola PJ, et al.. In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization. Nucleic Acids Res., 2015, 43: 8638-8650

[34] Scharner J, et al.. Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides. Nucleic Acids Res, 2020, 48: 802-816

[35] Crooke ST, Wang S, Vickers TA, Shen W, Liang XH. Cellular uptake and trafficking of antisense oligonucleotides. Nat. Biotechnol., 2017, 35: 230-237

[36] Gagnon KT, Corey DR. Guidelines for experiments using antisense Oligonucleotides and double-stranded RNAs. Nucleic Acid Ther., 2019, 29: 116-122

[37] Li Y, et al.. Comprehensive analysis of circRNA expression pattern and circRNA-miRNA-mRNA network in oral squamous cell carcinoma. Oral. Oncol., 2021, 121: 105437

[38] Qi T, et al.. Genetic control of RNA splicing and its distinct role in complex trait variation. Nat. Genet., 2022, 54: 1355-1363

[39] Hoffman GE, Schadt EE. variancePartition: interpreting drivers of variation in complex gene expression studies. BMC Bioinforma., 2016, 17 483

[40] Stegle O, Parts L, Piipari M, Winn J, Durbin R. Using probabilistic estimation of expression residuals (PEER) to obtain increased power and interpretability of gene expression analyses. Nat. Protoc., 2012, 7: 500-507

[41] Ongen H, Buil A, Brown AA, Dermitzakis ET, Delaneau O. Fast and efficient QTL mapper for thousands of molecular phenotypes. Bioinformatics, 2016, 32: 1479-1485

[42] Cingolani P, et al.. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly, 2012, 6: 80-92

[43] Bernstein BE, et al.. The NIH Roadmap Epigenomics Mapping Consortium. Nat. Biotechnol., 2010, 28: 1045-1048

[44] Zhao W, et al.. POSTAR3: an updated platform for exploring post-transcriptional regulation coordinated by RNA-binding proteins. Nucleic Acids Res., 2022, 50: D287-D294

Funding

National Natural Science Foundation of China (National Science Foundation of China)(81672678)

the Key Project of Health Commission of Jiangsu Province (ZDB202001), the Project of Health Commission of Jiangsu Province (H2019034), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, 2018–87)

RIGHTS & PERMISSIONS

The Author(s)