Abstract LB-186: Development and validation of a novel RNA in situ hybridization assay to detect RNA polymerase I activity in vivo

Abstract

Abstract Increased ribosome biogenesis is a critical process requisite for the cancer cell phenotype as it is required to doulbe protein levels for cell replication. Ribosome synthesis is a complex process that is stimulated by a number of well-studied oncogenic pathways (MYC, mTOR, RAS-RAF-ERK) and repressed by tumor suppressors (pRb, p53, PTEN, p14/ARF). A key first step in ribosome biogenesis is the increased synthesis by RNA Polymerase I (Pol I) of the precursor rRNA, referred to as 45S rRNA, that is later processed into the 28S, 5.8S and 18S mature rRNA forms. This process is compartmentalized into the nucleolus. As such, the pharmacological inhibition of RNA Pol I is becoming a potentially important therapeutic strategy in cancer (PMIDs 20055700, 23953479, 24434211). A validated assay that can reflect relative levels of Pol I activity in routinely obtained tissue specimens would be a useful tool. For example it would allow the pretreatment screening (as a predictive biomarker) for those tumors likely to respond to Pol I inhibition, and, could also serve as a pharmacodynamic biomarker of Pol I inhibition. The 5’ external transcribed spacer (5’ETS) of the nascent precursor 45S transcript is rapidly removed and degraded; thus, its levels can be used as a surrogate for new rRNA transcription. We employed branched DNA amplified RNA in situ hybridization using probes developed with Advanced Cellular Diagnostics (ACD RNAscope 2.0). The 5’ ETS/45S CISH signal was restricted to nucleoli. The signal was markedly attenuated in cell lines and in formalin fixed paraffin embedded (FFPE) prostate tissue slices after pharmacological inhibition of RNA Pol I using BMH-21 or actinomycin D, demonstrating validity as a measure of RNA Pol I transcriptional activity. In clinical human prostate FFPE tissue sections and TMAs there was a marked increase in the signal in the presumptive precursor lesion (high grade prostatic intraepithelial neoplasia/HGPIN, n = 25) and in invasive adenocarcinoma lesions (n = 72, Kruskal-Wallis, p = 0.0001 and p = 0.0001, respectively) compared with normal luminal epithelium. These results are consistent with prior work using RT-PCR showing an increase in 5’ ETS/45S rRNA levels in prostate cancer tissues. In adenocarcinomas, the increase in 5’ ETS/45S signal was present throughout all Gleason scores and pathological stages at radical prostatectomy, with no marked difference among these. We developed and analytically validated a chromogenic in situ hybridization (CISH) protocol using RNAscope for detecting the 5’ external transcribed spacer (ETS) of the precursor 45S ribosomal RNA in FFPE human tissues. This novel assay was used to show increased rRNA transcription in human clinical samples of PIN and carcinoma, and should prove useful for detection of increased rRNA production in various tumor types and as a novel pharmacodynamic biomarker in clinical trials of RNA Pol I inhibitors. Citation Format: Gunes Guner, Paul Sirajuddin, Qizhi Zheng, Hester Liu, Ibrahim Kulac, Marikki K. Laiho, Angelo M. De Marzo. Development and validation of a novel RNA in situ hybridization assay to detect RNA polymerase I activity in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-186.