The Prostate · 2025
Use of Droplet Digital PCR for Consistent Detection of TMPRSS2:ERG Gene Fusion Transcripts Initiated In Vitro.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Check Megan H, Ernst Sarah E, Sfanos Karen S
Аннотация
BACKGROUND: Gene fusions are hybrid genes that arise from chromosomal rearrangements linking two independent genes. The most common gene fusion in prostate cancer involves the 5' androgen-regulated TMPRSS2 promoter fused with the 3' ETS transcription factor ERG. TMPRSS2:ERG (T:E) gene fusions occur in about half of all prostate cancers and are considered an early event in oncogenesis. Investigations into the mechanism behind T:E gene fusion initiation using in vitro systems are hindered by the technical limitations posed by fluorescence in situ hybridization and suboptimal sensitivity of reverse transcription quantitative PCR (RT-qPCR). The objective of this study was to develop a reliable, user-friendly method of detecting low-abundance T:E gene fusion transcripts as a read-out for putative T:E gene fusions generated in cells.
METHODS: We assessed the sensitivity of droplet digital PCR (ddPCR) by quantifying T:E gene fusion transcripts using gene fragment- or cell-based standard curves. Next, we evaluated dihydrotestosterone (DHT), genotoxic insults (irradiation, etoposide), and inflammatory agents tumor necrosis factor-alpha (TNF-α) and hydrogen peroxide (H2O2) as initiators of T:E fusions in a fusion-negative prostate cell line (LNCaP). Finally, we performed RT-qPCR to measure the expression of androgen receptor (AR), AR-regulated genes (TMPRSS2, NKX3.1) and the downstream activation target of TNF-α, NF-κB.
RESULTS: We identified ddPCR as a sensitive method for detecting rare T:E gene fusion transcripts and observed consistent detection in reactions containing a single T:E gene fragment, or 1 fusion-positive cell per 10,000 fusion-negative cells. Consistent with prior studies, the ddPCR assay identified DHT combined with etoposide as potent, synergistic initiators of T:E gene fusion transcript expression in LNCaP cells. We did not detect T:E gene fusion transcripts after LNCaP exposure to irradiation, TNF-α, or H2O2. We determined that TNF-α and H2O2 exposure led to global downregulation of AR signaling, which may have limited the formation or expression of treatment-initiated genomic T:E fusions. Therefore, one limitation of the ddPCR assay is the requirement for T:E fusion mRNA expression.
CONCLUSIONS: Our proposed method significantly improves the feasibility of testing novel initiators of the T:E gene fusion and can be applied to additional studies investigating mechanisms of gene fusion initiation in prostate and other cancers.
METHODS: We assessed the sensitivity of droplet digital PCR (ddPCR) by quantifying T:E gene fusion transcripts using gene fragment- or cell-based standard curves. Next, we evaluated dihydrotestosterone (DHT), genotoxic insults (irradiation, etoposide), and inflammatory agents tumor necrosis factor-alpha (TNF-α) and hydrogen peroxide (H2O2) as initiators of T:E fusions in a fusion-negative prostate cell line (LNCaP). Finally, we performed RT-qPCR to measure the expression of androgen receptor (AR), AR-regulated genes (TMPRSS2, NKX3.1) and the downstream activation target of TNF-α, NF-κB.
RESULTS: We identified ddPCR as a sensitive method for detecting rare T:E gene fusion transcripts and observed consistent detection in reactions containing a single T:E gene fragment, or 1 fusion-positive cell per 10,000 fusion-negative cells. Consistent with prior studies, the ddPCR assay identified DHT combined with etoposide as potent, synergistic initiators of T:E gene fusion transcript expression in LNCaP cells. We did not detect T:E gene fusion transcripts after LNCaP exposure to irradiation, TNF-α, or H2O2. We determined that TNF-α and H2O2 exposure led to global downregulation of AR signaling, which may have limited the formation or expression of treatment-initiated genomic T:E fusions. Therefore, one limitation of the ddPCR assay is the requirement for T:E fusion mRNA expression.
CONCLUSIONS: Our proposed method significantly improves the feasibility of testing novel initiators of the T:E gene fusion and can be applied to additional studies investigating mechanisms of gene fusion initiation in prostate and other cancers.