Novel Assay to Detect RNA Polymerase I Activity In Vivo...utilize a branched DNA ampliﬁcation...

Metabolism

Novel Assay to Detect RNA Polymerase IActivity In VivoGunes Guner1, Paul Sirajuddin2, Qizhi Zheng1, Baoyan Bai2, Alexandra Brodie2,Hester Liu2, Taija af H€allstr€om3, Ibrahim Kulac1, Marikki Laiho2,4, andAngelo M. De Marzo1,4

Abstract

This report develops an analytically validated chromogenic insitu hybridization (CISH) assay using branched DNA signalamplification (RNAscope) for detecting the expression of the 50

external transcribed spacer (ETS) of the 45S ribosomal (r) RNAprecursor in formalin-fixed and paraffin-embedded (FFPE)human tissues. 50ETS/45S CISH was performed on standardclinical specimens and tissue microarrays (TMA) from untreatedprostate carcinomas, high-grade prostatic intraepithelial neopla-sia (PIN), and matched benign prostatic tissues. Signals werequantified using image analysis software. The 50ETS rRNA signalwas restricted to the nucleolus. The signal was markedly attenu-ated in cell lines and in prostate tissue slices after pharmacologicinhibition of RNA polymerase I (Pol I) using BMH-21 or actino-mycin D, and by RNAi depletion of Pol I, demonstrating validityas a measure of Pol I activity. Clinical human prostate FFPE tissue

sections and TMAs showed a marked increase in the signal in thepresumptive precursor lesion (high-grade PIN) and invasive ade-nocarcinoma lesions (P ¼ 0.0001 and P ¼ 0.0001, respectively)compared with non-neoplastic luminal epithelium. The increasein 50ETS rRNA signal was present throughout all Gleason scoresand pathologic stages at radical prostatectomy, with no markeddifference among these. This precursor rRNA assay has potentialutility for detection of increased rRNA production in varioustumor types and as a novel companion diagnostic for clinicaltrials involving Pol I inhibition.

Implications: Increased rRNA production, a possible therapeutictarget for multiple cancers, can be detected with a new, validatedassay that also serves as a pharmacodynamic marker for Pol Iinhibitors. Mol Cancer Res; 15(5); 577–84. �2017 AACR.

IntroductionAn essential feature of tumor growth is the need for increased

ribosome biogenesis. This is required for protein synthesis tosupplement cellular proteins requisite for a number of cancer cellactivities including cellular replication. In keeping with this,abundant literature has shown that alterations in the size andnumber of nucleoli as measured by silver staining (AgNOR),which correlates with nucleolar transcriptional activity, are com-mon in many human cancers and are commonly found to be ofprognostic significance in different cancer types (1–4). The first

and rate-limiting step of ribosome biogenesis is transcription ofthe 47S precursor ribosomal RNA by RNA polymerase I (Pol I).Pol I transcription is controlled by three mechanisms: transcrip-tion initiation by preinitiation complex assembly determined bymetabolic, proliferation, and differentiation signals; transcriptionelongation determined by elongation and chromatin factors; andepigenetic marking of the active and inactive gene repeats (5, 6).Pol I transcription is activated by most known oncogenes andoncogenic pathways (such as MYC, mTOR, Akt/PKB, MEK/ERK),and suppressed by predominant tumor-suppressor pathways(such as p53, Rb, ARF, Pten; refs. 7–13). Given the highly frequentalterations of these pathways in human cancers, deregulation ofPol I is expected to be pervasive. However, only few studies haveused analytical tools to assess the overall levels of Pol I activity ormature rRNA levels in clinical specimens (14, 15).

Pol I transcription is compartmentalized into the nucleolusleading to the synthesis of a 13-kb (47S/45S) rRNA precursor thatis rapidly processed into the 28S, 5.8S, and 18S mature rRNAforms (5, 16). The 50 external transcribed spacer (50ETS) of theprecursor transcript is very short lived, and thus the levels can beused as a surrogate for rRNA transcription activity (16, 17).Notably, in actively growing cells, rRNA transcription constitutesup to 60% of total cellular transcription, making this process, andthe ensuing building of ribosomes, the most energy-consumingprocess in cells (18). Given the predicted deregulation of rRNAsynthetic activity in cancers, a number of groups have suggestedthat Pol I inhibitionmaybe a therapeutic target in cancer (7, 19). Anumber of new compounds and existing cancer drugs, such asactinomycin D and topoisomerase poisons, have been shown to

1Department of Pathology, Urology and Oncology, Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland. 2Department of Radiation Oncologyand Molecular Radiation Sciences, Johns Hopkins University School of Medicine,Baltimore, Maryland. 3Institute for Molecular Medicine Finland, University ofHelsinki, Helsinki, Finland. 4SidneyKimmel ComprehensiveCancer Center, JohnsHopkins University School of Medicine, Baltimore, Maryland.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Current address for B. Bai: Department of Immunology, Institute for CancerResearch, Oslo University Hospital HF, The Norwegian Radium Hospital, Oslo,Norway.

Corresponding Authors: Angelo M. De Marzo, Johns Hopkins University Schoolof Medicine, 1550 Orleans Street, Baltimore, MD 21231. Phone: 410-614-5686,Fax: 410-502-9817; E-mail: [email protected]; or M. Laiho, Phone: 410-502-9748; Fax: 410-502-2821; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0246

�2017 American Association for Cancer Research.

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intervene with Pol I transcription (20–22). In a recent study bymembers of our group, Peltonen and colleagues (23) identifiedBMH-21 as (i) a potent andnovel inhibitor of Pol I that resulted inmarkedly decreased levels of the 50ETS rRNA; (ii) an activator ofnucleolar stress leading to altered localization of nucleolar pro-teins; and (iii) an inducer of degradation of the Pol I catalyticsubunit RPA194.

Prostate cancer appears particularly well suited to assesschanges in Pol I transcription. For example, it has been knownfor decades that a key diagnostic feature in histopathologic tissuesections is an increased size (and number) of the nucleolus, andthis feature is present in nearly 100%of prostate cancers (24–26).More recent studies have shown that this increased size correlateswith increased levels of the 45S/50ETS aswell as 5.8S, 18S, and28SrRNA levels (15). Further, prostate cancers have frequent over-expression of MYC which regulates nucleolar size, and commonloss of PTEN, which can also activate Pol I transcription and effectnucleolar size and function (27–29).

Here, we describe the development of a novel in situ hybrid-ization–based assay for functional measurement of Pol I activityusing a probe targeting the 50ETS rRNA precursor transcript.Improved technologies for performing RNA in situ hybridizationhave been commercially introduced recently (30). These assaysrely on the simultaneous hybridization of a number of pairs ofadjacent probes (so called Z-pairs) for enhanced specificity andutilize a branched DNA amplification technique to increasesensitivity (30, 31). We apply here this assay to studies of Pol Iactivity in cancer cell lines and prostate cancer and benign tissuesto demonstrate its promise as amarker of rRNA synthesis and as anovel pharmacodynamic biomarker for monitoring inhibition ofPol I in vivo.

Materials and MethodsCell lines and treatments

A375 (CRL-1619) human melanoma cell line was obtainedfrom the American Type Culture Collection, maintained inDMEMwith 10% FBS, and was authenticated using short tandemrepeat analysis by the Johns Hopkins Genetic Resources CoreFacility and tested periodically for Mycoplasma using qPCR withnegative results.

For embedding of cultured cells to paraffin, approximately40 million cells were harvested, resuspended in 10% neutral-buffered formalin, and pelleted by gentle centrifugation on top of2% agarose in PBS (32). Fixed cell pellets were placed in histologycassettes and processed for paraffin embedding. Actinomycin Dwas obtained from Sigma. BMH-21was synthesized in-house andverified for purity by mass spectrometry and nuclear magneticresonance (23).

Prostate tissue culturesRadical prostatectomy specimens were cored and sliced at 300

mmas detailed before (33, 34). The tissue sliceswere culturedwithRPMF-4A medium (35) supplemented with growth factors in ahumidified tissue culture incubator at 37�C. Following treat-ments, tissue slices were fixed with 10% formalin, embedded inparaffin, and cut to 4 mm. Prostate tissues were obtained withapproval of the Institutional Review Boards of the Johns HopkinsMedical Institutions (Baltimore, MD) and theHelsinki UniversityCentral Hospital (Helsinki, Finland).

ImmunohistochemistrySections were deparaffinized, rehydrated, and antigen retrieval

was conducted in citrate buffer at pH 6.1 (Dako). Slides wereblocked with 3% hydrogen peroxide and incubated with primaryantibodies overnight at 4�C. The slides were stained with Power-Vision detection system (Leica Biosystems) for 30 minutes atroom temperature followed by development with DAB solution(Invitrogen). Nuclei were counterstained with hematoxylin, andslides were visualized under brightfield microscope (NikonEclipse 50i) and imaged using Nikon DS-Fi1 color CCD camera.The following primary antibodies and dilutions were used:mouse anti-RPA194 (1:10,000; Santa Cruz Biotechnology), NPM(1:80,000; Abcam), and NCL (1:10,000; Abcam).

RT-qPCRRNA was isolated using TRIzol, and RT-qPCR was conducted

essentially as in ref. 23. Analyses were normalized against GAPDHand conducted using the DDCt method. Primer sequences for50ETS were GAACGGTGGTGTGTCGTT (forward) and GCGTCT-CGTCTCGTCTCACT (reverse).

In situ hybridizationIn situ hybridization for 50ETS precursor rRNA using locked

nucleic acid (LNA) probe was conducted as previously described(36, 37). Digoxigenin-labeled 50ETS LNA probe (GACGTCACCA-CATCGATCGAAG) was from Exiqon. The cells were counter-stained for DNA using Hoechst 33258 and mounted in VECTA-SHIELD mounting medium (Vector Laboratories). Images werecaptured using Axioplan2 fluorescence microscope (Zeiss)equipped with AxioCam HRc CCD-camera and AxioVision 4.5software using EC Plan-Neofluar 40x/0.75 objective (Zeiss).

50ETS/45S ISH assayChromogenic in situ hybridization (CISH) studies demon-

strating the presence of 50ETS/45S rRNA were performedaccording to the manufacturer's instructions (ACD RNAscope2.0 Brown Kit). FFPE slides were baked at 60�C for 1 hour, thendeparaffinized with exposure to xylene twice, 10 minutes eachtime, followed by stirring in 100% ethanol twice and air-drying, then rehydration with distilled H2O for 2 minutes.Pretreatment solution 1 was applied to the slides for 10minutes at room temperature. The slides were then boiled inpretreatment solution 2 at 100�C for 15 minutes, followed byprotease digestion in pretreatment solution 3 for 30 minutes at40�C to allow target accessibility. ACD target probe (Hs-45SrRNA, # 410401, 1/10 for human tissues, 1/40 for cell lines)was applied, and the slides were incubated in a HybEZ TMOven at 40�C for 2 hours. Slides were washed twice with 1Xwash buffer for 2 minutes at room temperature. Signal ampli-fication steps were as follows: amplification reagents 1 and 3were incubated for 30 minutes, amplification reagents 2 for 15minutes; amplification steps 1, 2, and 4 took place in the ovenat 40�C. Slides were washed with the 1x wash buffer betweeneach amplification step. DAB solution was applied for 10minutes at room temperature, and the slides were washedwith distilled H2O. Fifty percent Gill's hematoxylin wasapplied for 2 minutes for counterstaining, and the slides wererinsed in 0.01% ammonia for 10 seconds. Slides were passedthrough 100% ethanol, then xylene and mounted with Cyto-seal mounting medium.

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Whole-section slides and image analysisNine standard slide/whole-section slides of different prosta-

tectomy specimens were used for the demonstration of 50ETS/45Ssignal in different lesions. 50ETS/45S and hematoxylin–eosin-stained slides were scanned with a 40x objective using the Aperioimaging system (Leica Biosystems), annotated, and the annota-tions were analyzed using the Aperio ImageScope software (LeicaBiosystems). Regions of atrophy, normal appearing benign pros-tatic epithelium, high-grade prostatic intraepithelial neoplasia(PIN), and carcinoma foci were annotated as separate regions.The ratio of the number of positive (brown) pixels over the totalnumber of nuclear pixels (brown þ blue) was calculated perannotation.

Patient selection, TMA construction, and image analysisTissues were obtained from untreated patients undergoing

radical retropubic prostatectomy (RRP). These 66 radical prosta-tectomies obtained at Johns Hopkins Hospital (Baltimore, MD)were used for the construction of three tissue microarrays (TMA)as described (38). All patients were treated by RRP in 2012 to2014, and the patient ages ranged from 41 to 71 (mean, 59.31;median, 60), and the race of the patients were 82% white (52patients), 9% African American (6 patients), and the rest wereunknown. Per patient, the area of tumor with the highest Gleasonscore was spotted 3 to 5 times for TMA construction with a0.6 mm core size. One to 5 spots of benign prostate per patientwere evaluable in each TMA. A spectrum of normal tissues wasspotted multiple times in each TMA as controls.

Both hematoxylin–eosin-stained and 50ETS/45S CISH assayslides were scanned at x40 using the Aperio imaging system andviewed using the TMAJ-FrIDA software (an ImageJ plugin; http://tmaj.pathology.jhmi.edu/; refs. 28, 39). Hematoxylin–eosinslides of TMAs were assessed by a pathologist (G. Guner) todetermine the presence/absence of prostate carcinoma, benignglands, atrophy, andhigh-grade PIN. AGleason scorewas given toeach spot of tumor, and areas of tumor were annotated by thepathologist using the TMAJ-FrIDA software. TMAJ-FrIDAwas usedto determine the number of pixels and the intensity of the pixels inthe annotated areas that had 50ETS/45S signal. The area of brownpixels over total annotation area (brown þ blue) was calculated.Means of these ratios per casewere used in analyses. Amedian of 5tumor cores was assessed per patient (mean, 5.5; range, 1–8). Thisstudy was approved by the Institutional Review Board of JohnsHopkins Medical Institutions (Baltimore, MD).

Statistical analysisThe Kruskal–Wallis test was used to compare the 50ETS/45S

signals between different diagnostic categories and between dif-ferent grades and stages of disease. STATA (version 14, StataCorp)was used for analysis.

Results50ETS/45S probe localizes exclusively to nucleoli, and its signalis markedly reduced by Pol I inhibition in cancer cells andhuman prostate tissues

Our earlier work has shown that BMH-21 profoundly inhibitsPol I transcription and nascent rRNA synthesis, and decreases thenucleolar abundance of RPA194, the Pol I large subunit. Todevelop probes for detection of nucleolar rRNAs, wefirst designeda LNA probe against the short-lived 50ETS rRNA precursor and

used the probe to detect changes in its abundance and localiza-tion. Costaining of RPA194was used tomark the nucleolus and todetect cellular responses to two Pol I inhibitors, BMH-21 andactinomycin D. The 50ETS signal perfectly colocalized with that ofRPA194 within nucleoli (Fig. 1). Following short (3 hours)incubation times with the drugs, the 50ETS signal was abolished,and RPA194wasmarkedly decreased by BMH-21. Actinomycin Dcaused the relocalization of RPA194 to nucleolar caps, a hallmarkfeature of Pol I transcription stress (Fig. 1; ref. 40). The changes inRPA194 andPol I activity are consistentwith thosewehave shownbefore (23). These findings document that the 50ETS precursorrRNA can be used as a marker for dynamic changes in Pol Itranscription.

Optimization of the conditions for 50ETS chromogenic in situhybridization (referred to as 50ETS/45S) was performed usingRNAscope technology relying on the manufacturer's recommen-dations with the only change relating to the dilution of the probeconcentration. The chosen probe locations on an rRNA copy areshown in Fig. 2A. We first tested the specificity of the probe usingthe cultured human A375 melanoma cell line. The cells were leftuntreated, or treated with BMH-21 for 3 hours. In addition, wetransfected the cells with siRNAs that target RPA194, the catalyticsubunit of Pol I, and which we have shown to decrease RPA194protein expression, inhibit nascent rRNA synthesis, and reducecancer cell growth (23). Figure 2B shows that the 50ETS/45Shybridization signal is localized exclusively within nuclei, withsignals showing a stereotypic nucleolar pattern. Treatment of thecells with BMH-21 or depletion of RPA194 using RNAi largelyabolished the 50ETS/45S signal.

To test the applicability of the probe for formalin-embeddedsamples, we cultured A375 cells in the presence of increasingconcentrations of BMH-21 for 3 hours followed by trypsinizingthe cells and fixing them in formalin followed by paraffin embed-ding (FFPE) to simulate human clinical tissue sections. Theparaffin-embedded specimens were then hybridized with the

Figure 1.

Colocalization of 50ETS rRNA precursor and Pol I. Cells were treated with orwithout BMH-21 (1 mmol/L) or actinomycin D (40 nmol/L) for 3 hours, fixed, andhybridized with a LNA probe against 50ETS rRNA precursor (green), stained forRPA194 (red), and counterstained for DNA (blue). Scale bar, 10 mm.

RNA Hybridization Assay for RNA Pol I Activity

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Figure 2.

Validation of 50ETS/45S CISH assay. A, rRNA locus and location of probes for CISH in the 50ETS. B, A375 cells were treated with BMH-21 (1 mmol/L) for 3 hours,or transfected with RPA194 siRNAs and incubated for 48 hours, followed by CISH for 50ETS rRNA. Original magnification, x400. C, A375 cells were treatedeither with vehicle (DMSO) or the indicated concentrations of BMH-21. Cells were pelleted and processed for paraffin blocks. Top section shows CISH for 50ETS/45S,and bottom section shows IHC for RPA194. The signals were quantified and are plotted as compared with the controls (right). r, Pearson correlation. Originalmagnifications, x200.D, Cellswere treatedwith increasing concentrations of BMH-21 for 3 hours, and the cellswere either processed for paraffin blocks for 50ETS/45SCISH, or RNA was isolated and analyzed by RT-qPCR using 50ETS rRNA primers. N ¼ 2 biological replicates for CISH assay, and N ¼ 3 biological replicates forRT-qPCR. Scale bar, 100 mm. E, The CISH signals were quantified using TMAJ-FriDA. RT-qPCR was normalized using the DDCt method against GAPDH. Datarepresent mean and SEM.

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50ETS precursor probe or stained for RPA194 using IHC. The50ETS/45S signal decreased in a concentration-dependentmannerin the BMH-21-treated samples and correlated with the reductionof staining in RPA194, as anticipated (Fig. 2C).

We further compared the 50ETS/45S CISH assay with themeasurement of Pol I transcription activity using RT-qPCR.Cells were treated with increasing concentrations of BMH-21,paraffin-embedded as above, and hybridized with the 50ETS/45S probe, or RNA was isolated and analyzed by RT-qPCR for50ETS rRNA. As shown in Fig. 2D and E, the 50ETS/45S signal byin situ hybridization and the levels of 50ETS rRNA bothdecreased in a BMH-21-dose dependent manner response. Thetwo assays also correlated with each other (Pearson correlationcoefficient ¼ 0.77, P ¼ 0.043; Supplementary Fig. S2). Theseapproaches demonstrate the specificity and overall linearity ofthe assay for the detection of active rRNA transcription and itsinhibition in FFPE sections.

We thenused the50ETS/45SCISHassay todetect Pol I activity inhuman prostate tissues derived from radical prostatectomiescultured ex vivo (33, 34). The prostate tissues were treated withor without BMH-21. The tissues were processed by FFPE andhybridized with the 50ETS/45S precursor probe, or stained withantibodies against nucleolar proteins NPM, NCL, and RPA194.NPM and NCL were included as well-established markers of Pol Itranscription stress that occurs in response to BMH-21 (23, 41).We found a marked reduction in 50ETS/45S signal by BMH-21 inthe tissue sections concomitant with the translocation of NPMand NCL from the nucleoli to the nucleoplasm, and reduction inthe abundance of RPA194 (Fig. 3A and B). This demonstrates the

potential of the 50ETS/45S CISH assay to function as a pharma-codynamic biomarker in human tissue sections.

50ETS/45SCISH signal ismarkedly increased in humanPIN andadenocarcinomas in FFPE sections

We next performed 50ETS/45S CISH on a number of prostatecancer tissues using standard sections (n ¼ 30 areas each ofcarcinoma and benign from 9 patients) from radical prostatec-tomy specimens. Hybridization signals were consistent withnucleolar localization in all cell types, and there was a clearincrease in signal in cancer cells and PIN cells as compared withnormal luminal cells (Table 1; Supplementary Fig. S1; Fig. 4).Quantification of these results using Aperio image analysis soft-ware showed a marked increase in staining area within PIN andcarcinoma cell nuclei (Table 1; P ¼ 0.0001, Kruskal–Wallis).

To get a more comprehensive look across primary prostatecancer specimens, we next performed CISH and quantitative

Table 1. 50ETS/45S signal positivity ratios among different diagnoses insections of whole-mount prostatectomies

Histologic type

Number of regionsannotated (numberof patients)

Mean 45Spositivity (SD) P valuea

Benign prostate epithelium 43 (7) 0.04 (0.003) —

Atrophy 87 (6) 0.03 (0.002) 0.1876PIN 13 (4) 0.12 (0.012) 0.0001Carcinoma 85 (9) 0.11 (0.011) 0.0001aP values showcomparison of 45S signalswith that of benign/normal epitheliumby Kruskal–Wallis.

Figure 3.

Suppression of 50ETS/45S CISH signal after BMH-21 treatment of prostate tissue slices. Tissue slices were treated with either vehicle or BMH-21 (2 mmol/L)for 24 hours, and fixed. A, Staining of tissue slices for nucleolar markers NPM, NCL, and RPA194. B, CISH for 50ETS/45S was performed on tissues after fixing informalin and paraffin embedding. Note marked reduction in CISH signals after BMH-21 treatment. Original magnifications, x400; insets represent 2.8-foldmagnifications.






image analysis on a number of TMAs (Fig. 5) fromRRP specimenscontaining benign epithelium, high-grade PIN, and invasiveadenocarcinoma. Supplementary Table S1 shows the breakdownof cases on the TMAs by Gleason score grade groups (42) and

pathologic stage, and Table 2 shows the number of overall TMAspots of each diagnostic category (normal, atrophy, PIN, andcarcinoma) as well as their median scores and P values as com-pared with benign. The results in carcinoma (P ¼ 0.0001 vs.

Figure 4.

50ETS/45S CISH in human prostatewhole tissue sections.A, Left plot showsbox plot results of quantification of (D)normal, (C) high-grade PIN, and (B)carcinoma regions (n ¼ 30). Originalmagnifications, x200. Boxes arebounded by 25th and 75th percentiles,with median shown as line. Whiskersare 2x the interquartile range, andoutliers are shown as individual datapoints. Y-axis shows the ratio of thearea of positive pixels over the area oftotal nuclear pixels (positivity).

Figure 5.

50ETS/45S CISH signals in TMA spots and quantification of region with FrIDA software. A, Image on left shows representative tumor spot, and top right showsthe whole TMA section at very low power. Region of interest in this TMA spot (tumor) is highlighted by the black area mask, and the nucleolar 50ETS/45S CISH signalmask is highlighted in red. Nuclei are highlighted in the blue mask. B, Box plot of quantified staining data from indicated diagnostic categories showing markedincrease in PIN and carcinoma. Bottom plots (C and D) show that signals were not quantitatively different across different Gleason score and pathologic stages.

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benign) are consistent with prior studies that have shown anincrease in levels of 45S rRNA in human prostate cancer speci-mens compared with matched normal specimens using qPCR(15). The result in PIN (P ¼ 0.0001 vs. benign) demonstrates forthe first time the apparent increase in levels of Pol I activity in thislesion in humans in vivo. Quantification of 50ETS/45S CISHsignals across histologic grade by Gleason score, or by pathologicstage at radical prostatectomy, showed that the levels were con-sistently increased in all cancer grades and stages, with no signif-icant differences among the various groups (Table 2 and Fig. 5).

DiscussionThe pharmacologic inhibition of RNA Pol I is becoming a

potentially important therapeutic strategy in cancer (7, 19, 23).The therapeutic index using Pol I inhibition relies at least in parton the fact that tumor cells are often found to be in an anabolicstate, with higher levels of ribosome biosynthesis than theircorresponding normal counterparts (7, 9, 43). A validated assaythat can determine whether there is indeed an increase in Pol Iactivity in routinely obtained tissue specimens, as well as asimilarly applicable pharmacodynamic marker of target inhibi-tion, would be a valuable companion diagnostic for such ther-apeutic trials.

We developed a CISH assay to examine the level of expressionof the 50ETS/45S rRNA gene in routinely processed human clinicaltissue FFPE specimens. In contrast to the highly stable 18S/28SrRNA, the 50ETS region is processed cotranscriptionally and isshort-lived, and hence well suited for use as a marker for Pol Itranscription activity (17). As of now, we are not aware of altera-tions in the 50ETS processing rate by the cancer cells that mightaffect the abundance of the 50ETS transcript. The specificity andgeneral linearity of the assay were confirmed by pharmacologictreatments of cells and in vitro cultivated prostate tissue speci-mens, as well as, by the exclusive nucleolar localization of thesignal. Further, the 50ETS/45S signal was abolished by depletionof the Pol I catalytic subunit. Prior studies have shown increased50ETS/45S in prostate cancer tissue, as compared with matchedbenign tissues, using real-time RT-PCR (15). Thus, the presentresults showing increased levels of 50ETS/45S are consistent withthese findings but extend the applicability with ease of detectionin routine FFPE specimens. The precise method of quantificationof the signal (e.g., computerized image analysis vs. more tradi-tional visual estimations as in HER2/Neu IHC and FISH in breastcancer) that may be predictive of response to Pol I inhibitiontherapies, or that may serve as a pharmacodynamics marker inclinical FFPE specimens awaits additional clinical validationstudies.

We have recently found that tissue block age has a major effecton RNA in situ hybridization assays resulting in markedly

decreased signals, starting as soon as 1 year of age (J. Baena, Q.Zheng, A.M. De Marzo, manuscript in process) and thus wereprecluded from examining whether 50ETS/45S levels are associ-ated with poor outcome overall. Although we did not find acorrelation between pathologic risk factors of poor outcome and50ETS/45S levels, it was clear that in primary prostate cancer, levelswere increased through all grades and disease stages.

Pol I transcription is the rate-limiting step in ribosomebiogenesis and is activated by many prominent oncogenicdrivers such MYC, Akt/PKB, mTOR, and ERBB2 (7–13). Becausea number of pathways commonly altered in many differentcancers also result in increased rRNA synthesis, it is likely thatenhanced rRNA transcription will be a common finding inmultiple different cancer types. Surprisingly, despite the globalefforts of cataloguing cancer genome-wide changes, the contri-bution of Pol I transcription program to the cancer phenotypehas not been captured. No systematic approaches have beenimplemented for the detection of alterations in ribosomal RNAsynthesis in human cancers. With the assay described here,these analyses now become possible and facilitate the probingof Pol I activity in diverse cancer types using routine FFPEsections and TMAs.

Overexpression of rRNA levels, including increased transcrip-tion and processing, may be explained at least in part by the well-known common overexpression of MYC in human PIN andprostate cancer as well as MYC's known function in regulatingPol I and a number of nucleolar related genes (28, 29, 44). In fact,overexpression of MYC in the mouse prostate luminal epithelialcells results in highly prominent nucleolar size enlargement andnumerical increase, as well as increases in rRNA levels, including45S rRNA (29, 44).

In summary, we describe here a simple, robust, and sensitiveassay for the detection rRNA synthesis applicable to human cancerspecimens. The assay should be useful for the broad analyses ofchanges in rRNA transcription in human cancers and monitoringbiodynamic treatment responses in Pol I–targeted preclinical andclinical trials.

Disclosure of Potential Conflicts of InterestM. Laiho has ownership interest in a patent on BMH-21. No potential

conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G. Guner, M. Laiho, A.M. De MarzoDevelopment of methodology: G. Guner, P. Sirajuddin, H. Liu, M. Laiho,A.M. De MarzoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):G. Guner, P. Sirajuddin, B. Bai, H. Liu, T. af H€allstr€om,I. Kulac, A.M. De MarzoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Guner, P. Sirajuddin, H. Liu, M. Laiho, A.M. DeMarzoWriting, review, and/or revision of the manuscript: G. Guner, P. Sirajuddin,M. Laiho, A.M. De MarzoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Guner, Q. Zheng, A.M. De MarzoStudy supervision: G. Guner, M. Laiho, A.M. De Marzo

Grant SupportThis study was supported by NIH 1R01CA172069, NIH R21CA193637,

2014 Safeway Foundation-PCF Challenge Award, Harrington Scholar-Innova-tor Award, Academy of Finland (288364; to M. Laiho), NIH P30 CA006973 (toM. Laiho and A. De Marzo) and 2014 Safeway Foundation-PCF Challenge

Table 2. Histopathology of TMA cores and their 45S signal positivity ratios

Histologic type

Number of coresannotated (numberof patients)

Mean 45Spositivity (SD) P valuea

Benign prostate epithelium 165 (60) 0.04 (0.007) —

Atrophy 28 (17) 0.04 (0.006) 0.5572Total PIN 24 (21) 0.07 (0.005) 0.0001Carcinoma 352 (64) 0.08 (0.004) 0.0001aP values show comparison of 45S signals with that of benign epithelium byKruskal–Wallis.






Award, NIH/NCI Prostate SPORE P50CA058236, the Department of DefenseProstate Cancer Research Program, DODAwardNos.W81XWH-10-2-0056 andW81XWH-10-2-0046 PCRP Prostate Cancer Biorepository Network (PCBN; toA. De Marzo).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 20, 2016; revised December 8, 2016; accepted January 2, 2017;published OnlineFirst January 23, 2017.

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Guner et al.




2017;15:577-584. Published OnlineFirst January 23, 2017.Mol Cancer Res Gunes Guner, Paul Sirajuddin, Qizhi Zheng, et al.

In VivoNovel Assay to Detect RNA Polymerase I Activity

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Novel Assay to Detect RNA Polymerase I Activity In Vivo...utilize a branched DNA ampliﬁcation...

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