Combined Transcriptomic and Epitranscriptomic Profiling Identifies THBS1 as A Regulator of Enzalutamide Resistance in Prostate Cancer

Cancer drug resistance arises not only from selection of resistant clones, but also through rapid activation of adaptive transcriptional programs. One mechanism of transcriptional regulation involves N6-methyladenosine (m⁶A) RNA modification, which dynamically regulates mRNA processing and alternative splicing, ultimately impacting cell fate and differentiation. In prostate cancer (PC), resistance to systemic therapies such as the androgen receptor pathway inhibitor (ARPI) enzalutamide is associated with a host of well-documented androgen receptor (AR) alterations, including amplification, mutation, and alternative splicing. Given these functions, we hypothesized that m⁶A modifications play a role in the transition to enzalutamide resistance in PC. To test this, we used methyl-RNA-immunoprecipitation followed by sequencing (MeRIP-seq) in parallel with RNA-seq to identify gene transcripts that were both differentially methylated and differentially expressed between enzalutamide-sensitive and enzalutamide-resistant PC cells. We filtered and prioritized these genes using clinical and functional database tools, including Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA), The Cancer Genome Atlas (TCGA), and the Oncology Research Information Network (ORIEN) avatar. Using this approach, we identified 487 transcripts that were both differentially methylated and differentially expressed and validated six of the top 12 candidates via targeted qPCR and MeRIP-PCR. One of these, <i>THBS1</i>, was found to have increased m⁶A level associated with decreased transcript levels in enzalutamide-resistant cells, a finding recapitulated in publicly available preclinical and clinical data. Moreover, in enzalutamide-sensitive cells, depletion of <i>THBS1</i> by siRNA-knockdown induced resistance to enzalutamide. While <i>THBS1</i> has previously been implicated in aggressive PC phenotypes, we now show that <i>THBS1</i> downregulation directly contributes to a rapid transition to enzalutamide resistance, suggesting a novel role for this gene in PC hormonal therapy resistance. These results constitute the first comprehensive epitranscriptomic profiling of ARPI resistance and identify <i>THBS1</i> as a potential driver of acute resistance in prostate cancer.