Recurrent read-through fusion transcripts in breast cancer

Abstract

Read-through fusion transcripts that result from the splicing of two adjacent genes in the same coding orientation are a recently discovered type of chimeric RNA. We sought to determine if read-through fusion transcripts exist in breast cancer. We performed paired-end RNA-seq of 168 breast samples, including 28 breast cancer cell lines, 42 triple negative breast cancer primary tumors, 42 estrogen receptor positive (ER+) breast cancer primary tumors, and 56 non-malignant breast tissue samples. We analyzed the sequencing data to identify breast cancer associated read-through fusion transcripts. We discovered two recurrent read-through fusion transcripts that were identified in breast cancer cell lines, confirmed across breast cancer primary tumors, and were not detected in normal tissues (SCNN1A-TNFRSF1A and CTSD-IFITM10). Both fusion transcripts use canonical splice sites to join the last splice donor of the 5' gene to the first splice acceptor of the 3' gene, creating an in-frame fusion transcript. Western blots indicated that the fusion transcripts are translated into fusion proteins in breast cancer cells. Custom small interfering RNAs targeting the CTSD-IFITM10 fusion junction reduced expression of the fusion transcript and reduced breast cancer cell proliferation. Read-through fusion transcripts between adjacent genes with different biochemical functions represent a new type of recurrent molecular defect in breast cancer that warrant further investigation as potential biomarkers and therapeutic targets. Both breast cancer associated fusion transcripts identified in this study involve membrane proteins (SCNN1A-TNFRSF1A and CTSD-IFITM10), which raises the possibility that they could be breast cancer-specific cell surface markers.

Fig. 1

Breast cancer associated read-through fusion transcripts. Two breast cancer associated read-through fusion transcripts, SCNN1A-TNFRSF1A (a) and CTSD-IFITM10 (b), were detected in paired-end RNA-seq performed on breast cancer cell lines and primary tumors and were not detected in a variety of non-neoplastic human tissues. The 5′ gene partner is depicted in green, and the 3′ gene partner is depicted in red. The fusion transcripts use endogenous splice sites to fuse the two transcripts and the angled black lines indicate which exons flank the fusion junction to result in the chimeric transcript. RNA-seq reads that span the fusion junction are depicted above the gene models and the sequence from the 5′ partner is in green text and the sequence from the 3′ partner is in red text. The intergenic chromosomal distance between the fusion partners is denoted in kilobase pairs (kbp). Breast cancer cell line cDNA was PCR amplified using primers in the distal ends of the partner genes, and clones were sequenced. The alignment of the cDNA to the genome and the canonical gene models at this locus are depicted for SCNN1A-TNFRSF1A (c) and CTSD-IFITM10 (d). Both fusion transcripts include all of the canonical exons and splice sites of the partner genes up to the fusion junction and the fusion junction maintains the reading frame of the canonical transcripts

Fig. 2

Expression of genes involved in breast cancer associated read-through fusion transcripts. a We computed the fraction of reads that include sequence from the fusion transcript rather than the un-fused canonical transcript. The fraction of fusion transcript reads for 5′ fusion partners are indicated in green, and the 3′ fusion partners are denoted in red for each of the samples. Mean and standard error of the mean are depicted in black. Less than 20 % of the 5′ fusion partners’ transcripts include the fusion sequence, indicating that most of the transcripts from the 5′ gene are not fused. A significantly larger fraction of the 3′ gene’s transcripts contain the fusion sequence (Mann–Whitney test: SCNN1A vs TNFRSF1A p = 0.0247, and CTSD vs IFITM10 p < 0.0001). b There is not a significant difference in the expression levels of the 5′ fusion partner between samples with or without the read-through fusion transcript (labeled fused and not fused, respectively). This indicates that increased expression of the 5′ fusion partner is not sufficient to induce read-through fusion transcripts that include the 3′ gene

Fig. 3

Western blots of breast cancer associated fusion proteins. We performed Western blots using antibodies raised to one of the fusion partner proteins for the breast cancer associated fusion transcripts. For each candidate fusion, we ran cell lysates from two cell lines with RNA-seq reads spanning the fusion junction and one cell line without RNA-seq reads spanning the fusion junction. In each blot, the canonical/native size of the targeted protein was detected in each cell line, and a band at the predicted fusion protein size was detected in the cell line with the most RNA-seq fusion-spanning reads (CTSD-IFITM10 in MCF-7, and SCNN1A-TNFRSF1A in HCC1954). A band corresponding to the size of the predicted fusion protein was also detected in the cell line with the second most RNA-seq fusion transcript reads for the SCNN1A-TNFRSF1A fusion (SUM-102). None of the cell lines without RNA-seq evidence of the fusion transcript produced fusion protein-sized bands

Fig. 4

CTSD-IFITM10 read-through fusion transcript siRNA knockdown. a We designed qPCR primers to flank the fusion junction of the CTSD-IFITM10 read-through fusion transcript and we designed two custom siRNAs to target the fusion junction. The sequence from the CTSD (the 5′ gene) is indicated in green and the sequence from IFITM10 (3′ gene) is indicated in red. The MCF7 breast cancer cell line was transfected with two siRNAs targeting the CTSD-IFITM10 fusion junction. b qPCR of the fusion transcript was performed 48 h after transfection. Both siRNAs significantly reduced the abundance of the fusion transcript relative to the controls, which included a non-targeting siRNA and a mock transfection that did not contain any siRNA. c A quantitative cell proliferation assay was performed 72 h after transfection. Both siRNAs significantly reduced the number of live cells relative to the controls