Abstract
Disclosure: B.J. Petri: None. B.L. Valdes: None. K.M. Piell: None. E.C. Rouchka: None. C.M. Klinge: None.
Acquired resistance to endocrine therapies (ET) in patients with estrogen receptor α (ER+) breast tumors results in metastatic spread. RNA modifications including N6-methyladenosine (m6A) play crucial roles in the post-transcriptional regulation of gene expression and have been implicated in cancer progression. We hypothesized that m6A epitranscriptomic alterations are associated with pathways in ET resistance. Direct-RNA sequencing technology (nanopore) was used to detect and map m6A modifications at single-nucleotide resolution to comprehensively profile m6A modifications in ET-resistant LCC9 and ET -sensitive MCF-7 breast cancer cell lines with or without 4-hydroxytamoxifen (4-OHT) treatment. Additionally, MCF-7 cells were treated with the METTL3 inhibitor STM2457 to identify m6A positions and transcripts directly regulated by METTL3 activity. We developed a new statistical method by integrating m6Anet, an existing 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). We identified 61 transcripts with DMR between vehicle-treated LCC9 compared to MCF-7 cells and 323 transcripts with DMRs between 4-OHT-treated LCC9 and MCF-7 cells. Fewer transcripts showed DMR after 4-OHT treatment in MCF-7 (41) versus LCC9 cells (399). Thus, more changes in m6A DMRs were detected in response to 4-OHT in the ET-resistant LCC9 versus ET-sensitive MCF-7 cells with “translation elongation” as the top pathway in enrichment analysis. STM2457 treatment resulted in significant changes to DMRs in MCF-7 cells with or without 4-OHT treatment and altered gene expression patterns associated with “cytosolic ribosome” in enrichment analysis. We identified many genes with multiple m6A modifications, e.g., AMIGO2 with 14 m6A sites at METTL 3 target DRACH motifs in MCF-7 cells, compared to no m6A modification in AMIGO2 in LCC9 cells. Our findings reveal distinct m6A modification patterns in ET-resistant LCC9 breast cancer cells compared to their ET-sensitive parental MCF-7 cells and in response to 4-OHT, suggesting a potential role for epitranscriptomic alterations in the development of ET resistance.
Presentation: 6/3/2024