Integrative analysis of oncogenic fusion genes and their functional impact in colorectal cancer

Abstract

Background: Fusion genes are good candidates of molecular targets for cancer therapy. However, there is insufficient research on the clinical implications and functional characteristics of fusion genes in colorectal cancer (CRC).

Methods: In this study, we analysed RNA sequencing data of CRC patients (147 tumour and 47 matched normal tissues) to identify oncogenic fusion genes and evaluated their role in CRC.

Results: We validated 24 fusion genes, including novel fusions, by three algorithms and Sanger sequencing. Fusions from most patients were mutually exclusive CRC oncogenes and included tumour suppressor gene mutations. Eleven fusion genes from 13 patients (8.8%) were determined as oncogenic fusion genes by analysing their gene expression and function. To investigate their oncogenic impact, we performed proliferation and migration assays of CRC cell lines expressing fusion genes of GTF3A-CDK8, NAGLU- IKZF3, RNF121- FOLR2, and STRN-ALK. Overexpression of these fusion genes increased cell proliferation except GTF3A-CDK8. In addition, overexpression of NAGLU-IKZF3 enhanced migration of CRC cells. We demonstrated that NAGLU-IKZF3, RNF121-FOLR2, and STRN-ALK had tumourigenic effects in CRC.

Conclusion: In summary, we identified and characterised oncogenic fusion genes and their function in CRC, and implicated NAGLU-IKZF3 and RNF121-FOLR2 as novel molecular targets for personalised medicine development.

Fig. 1

Identification and validation of fusion genes in CRC. a Overview of fusion gene filtering process. b Cross-validation with deFuse and FusionMap algorithms prior to PCR validation. GFP algorithm was used as the first detection tool, and then deFuse and FusionMap algorithms were applied to the 25 genes. c RT-PCR validation. Sanger sequencing confirmed 24 out of 25 fusion genes. An arrow denotes a junction point of a fusion gene

Fig. 2

Schematic overview of mutation profiling. Mutual exclusivity of fusion genes with oncogenic mutations was represented, except TMOD3-MAPK6. The numbers on top designate each patient sample ID with fusion genes; 1, TMOD3-MAPK6; 2, TPM3-NTRK1; 3, NAGLU-IKZF3; 4, ATIC-UXS1; 5, TMPRSS2-PDE9A and TPM3-NTRK1; 6, BOK-ING5; 7, GTF3A-CDK8; 8, TRIM24-BRAF; 9, RNF121-FOLR2; 10, PTPRK-RSPO3; 11, RASA1-LOC644100; 12, TAF4-NDRG3; 13, ANAPC1-ZC3H8 and ZYG11A-GPX7; 14, BOD1-WWC1, RIMS3-SCMH1, STRN-ALK, and USP32-NSF; 15, OSBPL10-GADL1; 16, APC-COMMD10 and VTI1A-TCF7L2; 17, RAB1B-DPP3; 18, LMNA-NTRK1; 19, CHIC1-HDX and PTPRK-RSPO3

Fig. 3

Predictions of biological functions of fusion genes. a Gene expression of donor or acceptor genes. Only the gene expression data of patients with high expression of donor or acceptor genes were presented. Red circle depicts patient with each fusion gene. The X-axis represents 47 paired normal samples (sky blue) and 147 tumour samples (pink). The Y-axis represents fragments per kilobase million (FPKM). b Schematic protein structure of fusion genes. Red arrow indicates fusion site. Protein domain abbreviations are as follows: EP-C, ARF7 effector protein C-terminus; FM, filament; FN3, fibronectin type III; F_rec, folate receptor family; Fu, furin-like repeat and cysteine-rich; Ig, immunoglobulin I-set; MAM, meprin/A5/mu; NG, NAGLU tim-barrel; NG-N, NAGLU N-terminal; PK, protein kinase; PK_Tyr, protein tyrosine kinase; St, striatin family; SUP, APC tumour suppressor protein; TD, tropomodulin; TM, tropomyosin; TS, thrombospondin type 1

Fig. 4

Biological function test. a Cell proliferation in CRC cells. Each MTT assay was performed three times. Empty vector is pCMV6-myc-DDK, except RNF121-FOLR2, which used pcDNA3.1-V5-His B. *P < 0.05 compared to empty vector, determined by the Student’s t-test; ^#P < 0.05 compared to each acceptor gene expression vector, determined by the Student’s t-test. b Cell migration assay of NAGLU-IKZF3 was performed three times. Harvested cells were seeded in transwell 48 h after transfection. *P < 0.05 compared to empty vector, determined by the Student’s t-test