Integrated Nanopore and short-read RNA sequencing identifies dysregulation of METTL3- m6A modifications in endocrine therapy- sensitive and resistant breast cancer cells

Funct Integr Genomics. 2025 Jul 9;25(1):149. doi: 10.1007/s10142-025-01658-2.

Abstract

The role of epitranscriptomic changes in the development of acquired endocrine therapy (ET)- resistance in estrogen receptor α (ER) expressing breast cancer (BC) is unknown. We tested the hypothesis that inhibition of METTL3, the methyltransferase responsible for the mRNA modification N-6 methyladenosine (m6A), alters m6A modifications and differentially regulates the abundance of mRNA transcripts in ET-sensitive MCF-7 versus resistant LCC9 ER + human BC cells. Differential m6A modifications were identified using direct mRNA sequencing (DRS) performed on five replicates for each cell line ± 1 µM STM2457, a selective METTL3 inhibitor, using Nanopore MinION long read RNA-seq. Parallel short read Illumina RNA-seq quantified differential transcript abundance in the same samples. Selected results were validated by RT-qPCR, m6A-RIP-qPCR, reporter assays, and western blot analysis. Statistical analysis combined m6Anet, a machine-learning algorithm designed to call m6A modified bases, with a generalized linear model following a binomial distribution analysis to identify significant differential m6A modification ratios (DMR). Distinct METTL3 dependent m6A modification patterns in LCC9 and MCF-7 cells were observed in differentially expressed genes (DEG) associated with ET-resistance, including EEF1A2, ACTB, FLNA, PDIA6, AMIGO2, TPT1, XBP1, and CITED4. Select results were validated in additional ET-resistant BC cell lines. m6A-RIP-RT-qPCR validated specific m6A sites. We examined the proximity of m6A sites to estrogen receptor α (ER α)-mRNA binding sites reported in MCF-7 cells. ACTB, PDIA6, and XBP1 demonstrated a short-range proximity, with m6A sites located within 100 bp of ERα binding sites, suggesting a role for m6A in influencing ERα-mRNA binding. Our work provides a framework for integrating DRS and DEG omics data. Our results suggest a role for dysregulation of m6A modifications in pathways implicated in ET resistance in BC.

Keywords: Breast cancer; Endocrine-resistance; Epitranscriptome; M6A; Nanopore DRS.

MeSH terms

  • Adenosine* / analogs & derivatives
  • Adenosine* / metabolism
  • Breast Neoplasms* / drug therapy
  • Breast Neoplasms* / genetics
  • Breast Neoplasms* / metabolism
  • Cell Line, Tumor
  • Drug Resistance, Neoplasm* / genetics
  • Estrogen Receptor alpha / genetics
  • Estrogen Receptor alpha / metabolism
  • Female
  • Gene Expression Regulation, Neoplastic
  • Humans
  • MCF-7 Cells
  • Methyltransferases* / antagonists & inhibitors
  • Methyltransferases* / genetics
  • Methyltransferases* / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Sequence Analysis, RNA

Substances

  • METTL3 protein, human
  • Methyltransferases
  • Adenosine
  • N-methyladenosine
  • Estrogen Receptor alpha
  • RNA, Messenger
  • ESR1 protein, human