Pan-Cancer Analysis Reveals Recurrent BCAR4 Gene Fusions across Solid Tumors

Abstract

Chromosomal rearrangements often result in active regulatory regions juxtaposed upstream of an oncogene to generate an expressed gene fusion. Repeated activation of a common downstream partner-with differing upstream regions across a patient cohort-suggests a conserved oncogenic role. Analysis of 9,638 patients across 32 solid tumor types revealed an annotated long noncoding RNA (lncRNA), Breast Cancer Anti-Estrogen Resistance 4 (BCAR4), was the most prevalent, uncharacterized, downstream gene fusion partner occurring in 11 cancers. Its oncogenic role was confirmed using multiple cell lines with endogenous BCAR4 gene fusions. Furthermore, overexpressing clinically prevalent BCAR4 gene fusions in untransformed cell lines was sufficient to induce an oncogenic phenotype. We show that the minimum common region to all gene fusions harbors an open reading frame that is necessary to drive proliferation.

Implications: BCAR4 gene fusions represent an underappreciated class of gene fusions that may have biological and clinical implications across solid tumors.

Graphical abstract

Figure 1.

Pan-cancer discovery of BCAR4 gene fusions. A, Dot plot of recurrent gene fusions by number of patients across cancer types. Callout boxes list detected 5′ partners of known gene fusions. B, Left, Structure of expressed BCAR4 fusion transcripts with the 5′ gene fusion partners represented in various colors and BCAR4 represented in green. Fusions are sorted by descending prevalence. B, Right, Representation of patients across cancer types expressing BCAR4 fusion transcripts. Rows correspond to the various BCAR4 isoforms. A cell is colored blue if the patient expresses a particular BCAR4 fusion, and the intensity corresponds to the number of RNA-seq reads supporting evidence of the fusion expression. STAD, stomach adenocarcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; BRCA, breast invasive carcinoma; ESCA, esophageal carcinoma; OV, ovarian serous cystadenocarcinoma; SKCM, skin cutaneous melanoma; BLCA, bladder urothelial carcinoma; LUAD, lung adenocarcinoma; UCEC, uterine corpus endometrial carcinoma; CRC, colorectal cancer; PRAD, prostate adenocarcinoma.

Figure 2.

BCAR4 gene fusions alter cell-cycle and proliferation. Representative dot plots of EdU-DNA stain (FxCycle) flow cytometry analysis and quantification of G1, S, G₂–M cell populations after siRNA-mediated silencing of BCAR4 fusions in SNU308 (n = 3; A) or TUHR14TKB (n = 4; B) cells. A and B, Right, qRT-PCR analysis confirmed knockdown of BCAR4 fusions. Representative dot plots of EdU-DNA stain (FxCycle) flow cytometry analysis and quantification of G1, S, G₂–M cell populations overexpressing EV or gene fusion (L-B or Z-B fusion) in HME1 (n = 4; C) or MCF10a cells (n = 6; D). Bar graphs present normalized mean ± SEM. Paired ratiometric two-tailed t tests were performed. C and D, Right, Cell growth curve analysis of HME1 (n = 5) or MCF10a (n = 6) cells overexpressing EV, L-B, or Z-B fusions. Graphs present mean ± SEM. Paired two-tailed t tests were performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

BCAR4 encodes a small protein that alters cell-cycle and proliferation. A, Left, Schematics depicting the BCAR4 gene, its predicted open reading frame in exon 4, and the tryptic peptides of BCAR4 detected by mass spectrometry. A, Right, BCAR4 peptide support in lung adenocarcinoma (LUAD), uterine corpus endometrial carcinoma (UCEC), ovarian serous cystadenocarcinoma (OV), and breast invasive carcinoma (BRCA). Representative dot plots of EdU-DNA stain (FxCycle) flow cytometry analysis and quantification of G1, S, G₂–M cell populations of B, HME1 (n = 6) and C, MCF10a (n = 3) overexpressing EV, L-B, or mutant L-B (mut L-B) fusions. Bar graphs present normalized mean ±SEM. Paired ratiometric two-tailed t tests were performed. B and C, Right, Cell growth curve analysis of HME1 (n = 6) or MCF10a cells (n = 4) overexpressing EV, LB, or mut L-B. Graphs present mean ±SEM. Paired two-tailed t tests were performed. D, Western blot of FLAG-tagged BCAR4 ORF expression in HME1 and MCF10a BCAR4-fusion–overexpressing cells, n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.