Profile of chimeric RNAs and TMPRSS2-ERG e2e4 isoform in neuroendocrine prostate cancer

Abstract

Purpose: Specific gene fusions and their fusion products (chimeric RNA and protein) have served as ideal diagnostic markers and therapeutic targets for cancer. However, few systematic studies for chimeric RNAs have been conducted in neuroendocrine prostate cancer (NEPC). In this study, we explored the landscape of chimeric RNAs in different types of prostate cancer (PCa) cell lines and aimed to identify chimeric RNAs specifically expressed in NEPC.

Methods: To do so, we employed the RNA-seq data of eight prostate related cell lines from Cancer Cell Line Encyclopedia (CCLE) for chimeric RNA identification. Multiple filtering criteria were used and the candidate chimeric RNAs were characterized at multiple levels and from various angles. We then performed experimental validation on all 80 candidates, and focused on the ones that are specific to NEPC. Lastly, we studied the clinical relevance and effect of one chimera in neuroendocrine process.

Results: Out of 80 candidates, 15 were confirmed to be expressed preferentially in NEPC lines. Among them, 13 of the 15 were found to be specifically expressed in NEPC, and four were further validated in another NEPC cell line. Importantly, in silico analysis showed that tumor malignancy may be correlated to the level of these chimeric RNAs. Clinically, the expression of TMPRSS2-ERG (e2e4) was elevated in tumor tissues and indicated poor clinical prognosis, whereas the parental wild type transcripts had no such association. Furthermore, compared to the most frequently detected TMPRSS2-ERG form (e1e4), e2e4 encodes 31 more amino acids and accelerated neuroendocrine process of prostate cancer.

Conclusions: In summary, these findings painted the landscape of chimeric RNA in NEPC and supported the idea that some chimeric RNAs may represent additional biomarkers and/or treatment targets independent of parental gene transcripts.

Keywords: Chimeric RNA; Neuroendocrine prostate cancer; TMPRSS2-ERG.

Fig. 1

The discovery pipeline and landscape of chimeric RNAs in prostate cancer. A The pipeline for discovering prostate cancer chimeric RNAs. The CCLE prostate related cell sequencing data were used for analysis. After filtering out of “M/M” fusions, GTEx fusions and PrEC LH fusions, 864 chimeric RNAs remain. 457 prostate cancer biased chimeric RNAs were identified after UCSC confirmation. B Circos plot depicting all identified chimeric RNAs in each prostate cell line. C Distributions of chimeric RNAs from HSPC, CRPC and NEPC. Chimeric RNAs were categorized based on their fusion type, junction position, and fusion protein coding potential. D Venn diagram shows the overlapping and specific chimeric RNAs among HSPC, CRPC, and NEPC. E–G Gene ontology analyses of parental genes involved in chimeric RNAs specific for HSPC, CRPC, and NEPC

Fig. 2

Validation of chimeric RNAs. A Gel images of RT- PCR products of the 80 candidate chimeric RNAs, with red arrows pointing to the chosen bands for Sanger sequencing. B Sanger sequencing results of the validated chimeric RNAs, with red lines marking the junction sites. Forward primer was used for Sanger sequencing in FXYD2-DSCAML1, and reverse primer was used in TMPRSS2-ERG, EEF2-SLC25A42, and SNX13-ATP2C1. C Gel images of RT- PCR products of the 15 candidate chimeric RNAs, with red arrows pointing to the correct bands. PrEC: PrEC LH. Mix1: cDNA mix of LNCaP, C4-2, PC3 and DU145. Mix2: cDNA mix of NCI-H660 and LASCPC-01

Fig. 3

Examination of NEPC specificity for the chimeric RNAs. A Gel images of qRT- PCR products of SPSB4-PXYLP1 and RIPPLY2-CYB5R4, which are not exclusively expressed in NEPC cell lines. B Quantitative analysis of SPSB4-PXYLP1 and RIPPLY2-CYB5R4 in different cell lines. C Validation of 13 NEPC specific chimeric RNAs in NCI-H660 and LASCPC-01. Red arrows point to correct products. Yellow arrow points to a non-specific product in LASCPC-01. D The schematic diagrams of four chimeric RNAs. Blocks represent exons. Black lines represent introns or intergenic regions. Red lines (arrows) represent junction sites. ****p < 0.0001

Fig. 4

Characteristic of chimeric RNA TMPRSS2-ERG (e2e4) in TCGA. A Normalized expression of TMPRSS2-ERG (e2e4) in 52 pairs of PCa and normal margin samples from TCGA. B–C Normalized expression of parental TMPRSS2 and parental ERG in 52 pairs of PCa and normal margin samples from TCGA. D–E The correlation between chimeric TMPRSS2-ERG (e2e4) and its parental genes. F–H Recurrence-free survival analysis of TMPRSS2-ERG F, parental TMPPSS2 G, and parental ERG H base on their normalized read counts. ***p < 0.001, *p < 0.05

Fig. 5

Validation of the TMPRSS2-ERG (e2e4) in cell lines and clinical samples. A Primers were designed based on the different exons of parental genes. B–D Gel images of Touch-down PCR products of the full length of TMPRSS2-ERG (e2e4) in NCI-H660 B, tumor mix samples C and adjacent normal mix samples D. It should be mentioned that the shallow band of R12-3 in tumor mix samples and R12 in adjacent normal mix samples are both non-specific amplifications confirmed by Sanger sequencing. E The schematic diagram of TMPRSS2-ERG (e2e4) translation, and the translated amino acid sequence. The sequence of extra coding region of TMPRSS2 is highlighted in green, and the sequence of extra coding region of ERG is highlighted in yellow. F Validation of protein coding potential of e1e4 and e2e4 plasmids using anti-Flag antibody and anti-ERG antibody

Fig. 6

TMPRSS2-ERG (e2e4) promotes docetaxel resistance and accelerates neuroendocrine process of prostate cancer. A Representative images and histogram of migration assays using LNCaP and C4-2 cells overexpressing TMPRSS2-ERG e1e4 or e2e4. B–C The CCK8 assay was used to measure cell viability in LNCaP and C4-2 cells when e1e4 or e2e4 was overexpressed. D The CCK8 assay was used to determine IC50 of enzalutamide in LNCaP cells with e1e4 or e2e4 overexpression. E Colony formation assay tested cell viability in C4-2 cells after docetaxel treatment when e1e4 and e2e4 were overexpressed. F The CCK8 assay was used to determine IC50 of docetaxel in C4-2 cells with e1e4 or e2e4 overexpression. G Gel images of RT-PCR product of e1e4 or e2e4 after using various concentrations of docetaxel treatment. H Representative image of the Western blotting analysis of CHGA, NSE and SYP levels after e1e4 or e2e4 overexpression in LNCaP and C4-2 cells. ****p < 0.0001, ***p < 0.001.**p < 0.01