Novel Assay to Detect RNA Polymerase I Activity In Vivo

Abstract

This report develops an analytically validated chromogenic in situ hybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5′ external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin fixed and paraffin embedded (FFPE) human tissues. 5′ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMAs) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN) and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5′ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacological inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade prostatic intraepithelial neoplasia/PIN) and invasive adenocarcinoma lesions (p=0.0001 and p=0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5′ETS rRNA signal was present throughout all Gleason scores and pathological stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition.

Effective targeting of RNA polymerase I in treatment‐resistant prostate cancer

“…We have recently developed an in situ hybridization assay for assessing 45S precursor rRNA transcripts in adherent cells and paraffin‐embedded samples utilizing RNA probes specific for the 5′ETS region . We treated LNCaP and MR49F cells with DMSO or BMH‐21 (1 µM) for 24 hours and assessed the presence of 45S precursor rRNA transcript using this probe.…”

“…ACD RNAscope 2.0 Brown Kit (Advanced Cell Diagnostics, Newark, CA) was used for 45S rRNA chromogenic in situ hybridization. Hybridization was carried out as stated in the manufacturer's protocol as described earlier …”

“…28 To We have recently developed an in situ hybridization assay for assessing 45S precursor rRNA transcripts in adherent cells and paraffin-embedded samples utilizing RNA probes specific for the 5′ ETS region. 14 precursor rRNA transcript using this probe. We observed a prominent decrease in the 45S transcript signal ( Figure 2F).…”

“…While these therapies are initially effective, resistance and progression of disease often occur, underscoring the need to develop new treatments . Of note, Pol I is highly upregulated in prostate cancer and in advanced, metastatic disease . The increase in Pol I may result from activation of Myc, or other driver genes, such as protein kinase B, mammalian target of rapamycin, or mitogen‐activated protein kinase/extracellular signal‐regulated kinase signaling pathways that are commonly altered in prostate cancer .…”

“…12 Of note, Pol I is highly upregulated in prostate cancer and in advanced, metastatic disease. 13,14 The increase in Pol I may result from activation of Myc, or other driver genes, such as protein kinase B, mammalian target of rapamycin, or mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathways that are commonly altered in prostate cancer. [15][16][17] Furthermore, the loss of negative regulators of Pol I transcription including PTEN, p53, or retinoblastoma protein can further lead to overt activation of the Pol I transcription program.…”