Abstract
Aberrant alternative splicing (AS) is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. Breast cancer-associated AS events are known to be linked to disease progression, metastasis, and survival of breast cancer patients. To identify altered AS programs occurring in metastatic breast cancer, we perform a global analysis of AS events by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq). We demonstrate that, relative to low-metastatic, high-metastatic breast cancer cells show different AS choices in genes related to cancer progression. Supporting a global reshape of cancer-related splicing profiles in metastatic breast cancer we found an enrichment of RNA-binding motifs recognized by several splicing regulators, which have aberrant expression levels or activity during breast cancer progression, including SRSF1. Among SRSF1-regulated targets we found DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes show different DCUN1D5 isoform ratios with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels, which are ultimately associated with poor overall and relapse-free survival in breast cancer patients. Collectively, our results reveal global AS features of metastatic breast tumors, which open new possibilities for the treatment of these aggressive tumor types.
Highlights
Introduction published maps and institutional affilFollowing transcription, introns are removed from the pre-mRNA transcript and exons are joined to each other to produce the mature messenger RNA, a process known as splicing [1]
To identify pre-mRNAs undergoing abnormal alternative splicing (AS) regulation in high-metastatic breast cancer cells compared to low-metastatic cancer cells, we performed a RASL-seq using RNAs extracted from MDA-MB-231 and MCF7 cell lines, respectively
We identified 925 AS events—~59.4% of a total of 1558 identified—significantly altered in MDA-MB-231 cells compared to MCF7 cells, suggesting a striking difference in splicing programs between these two cell lines (Supplementary Table S2)
Summary
MCF7 [American Type Culture Collection (ATCC, HTB-22TM)], MDA-MB-231 (ATCC, HTB-26TM), T47D (ATCC, HTV-133) cells were cultured in RPMI medium (HyClone, Logan, UT, USA, Cat: SH30027.01) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA, Cat: SH30084.03), 2 mM glutamine (Gibco, Grand island, NY, USA, Cat: 35050-061), 1X penicillin-streptomycin (HyClone, Logan, UT, USA Cat: SV30010). (ATCC, HTB-122) cells were cultured in DMEM medium (HyClone, Logan, UT, USA, Cat: SH30243.01) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA, Cat: SH30084.03), 2 mM glutamine (Gibco, Grand island, NY, USA, Cat: 35050-061), 1X penicillin-streptomycin (HyClone, Logan, UT, USA Cat: SV30010). Green PCR (Qiagen, Hilden, Germany, Cat: 204145) using a LyghtCycler 480 (Roche, Basel, Switzerland), according to the manufacturer’s instructions using GAPDH, B2M, or RPLP0 as an internal control. Primers used in this study are listed in Supplementary Table S1
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