Recurrent readthrough fusion transcripts in breas

Authors: Katherine E Varley Jason Gertz Brian S Roberts Nicholas S Davis Kevin M Bowling Marie K Kirby Amy S Nesmith Patsy G Oliver William E Grizzle Andres Forero Donald J Buchsbaum Albert F LoBuglio Richard M Myers

Publish Date: 2014/06/15

Volume: 146, Issue: 2, Pages: 287-297

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Abstract

Readthrough 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 readthrough fusion transcripts exist in breast cancer We performed pairedend RNAseq 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 nonmalignant breast tissue samples We analyzed the sequencing data to identify breast cancer associated readthrough fusion transcripts We discovered two recurrent readthrough fusion transcripts that were identified in breast cancer cell lines confirmed across breast cancer primary tumors and were not detected in normal tissues SCNN1ATNFRSF1A and CTSDIFITM10 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 inframe fusion transcript Western blots indicated that the fusion transcripts are translated into fusion proteins in breast cancer cells Custom small interfering RNAs targeting the CTSDIFITM10 fusion junction reduced expression of the fusion transcript and reduced breast cancer cell proliferation Readthrough 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 SCNN1ATNFRSF1A and CTSDIFITM10 which raises the possibility that they could be breast cancerspecific cell surface markersFusion genes with oncogenic activity were first identified in hematologic malignancies where chromosomal translocations frequently join two genes that result in an aberrant protein product 1 2 These fused genes have been valuable prognostic markers and therapeutic targets 3 The therapeutic value of identifying fusion genes is exemplified by the development of selective inhibitors targeted to the ABL kinase involved in the BCR–ABL fusion that is present in 95  of patients with chronic myelogenous leukemia 1 2 4 Most recurrent fusion genes have been identified in leukemias lymphomas and soft tissue sarcomas where cytogenetic approaches to detect chromosomal aberrations using spectral karyotyping fluorescent in situ hybridization and flow cytometry have been developed 5 Cytogenetic approaches to detect fusion genes in the more common forms of cancer epithelial tumors are hampered by the poor chromosome morphology complex karyotypes and cellular heterogeneity that typify these tumors although it has been posited that fusion genes are likely drivers of oncogenesis in these tumors as well 3 5 6 Until recently the most prevalent recurrent fusion genes identified in breast cancer were the ETV6NTRK3 fusion in secretory breast carcinoma a rare subtype of infiltrating ductal carcinoma 7 and the MYBNFIB fusion in adenoid cystic carcinomas another rare form of breast cancer 8 Recently genomewide microarray profiling the whole genome sequencing and the whole transcriptome sequencing have made it possible to systematically identify fusion genes in solid tumors With these methods recurrent fusions that contribute to malignancy have been identified in prostate cancer eg TMPRSS2 fused to ETS family transcription factors 9 10 11 in lung cancer EML4–ALK 12 and in breast cancer MAST kinases fused to NOTCH family genes 13 New technologies and informatics approaches are enabling the identification of recurrent fusion genes in more common epithelial cancers that may serve as valuable biomarkers and drug targets 13 14 15 16 17 18 19In addition to fusion genes created by genomic rearrangements fusion transcripts created by cis and transsplicing of mRNA in the absence of a DNA rearrangements have been detected by sequencing cDNA clone libraries and performing RNAseq 20 These chimeric RNAs have been detected at low levels in expressed sequence tag EST libraries 21 22 23 and low levels across benign and malignant samples 6 20 24 One particularly prevalent class of chimeric RNAs involves adjacent genes in the same coding orientation that are spliced together to form an inframe chimeric transcript that spans both genes In the recent literature these have been referred to as readthrough gene fusions transcriptioninduced chimeras cotranscription of adjacent genes coupled with intergenic splicing CoTIS or conjoined genes Several of these readthrough fusion transcripts have been identified specifically in prostate cancer and are associated with cellular proliferation and disease progression 25 26 27 28 29 30 31 32 33 Recurrent readthrough transcripts have not yet been characterized in breast cancer We used pairedend RNAseq to identify two novel recurrent readthrough fusion transcripts associated with breast cancer and we used genomic DNA sequencing qPCR cDNA clone sequencing small interfering RNA siRNA knockdown and Western blots to further confirm and characterize these fusion transcripts

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