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. Author manuscript; available in PMC: 2018 Feb 7.
Published in final edited form as: J Pathol. 2017 Nov 14;244(1):11–24. doi: 10.1002/path.4980

MYC drives overexpression of telomerase RNA (hTR/TERC) in prostate cancer

Javier A Baena-Del Valle 1,2, Qizhi Zheng 1, David M Esopi 3, Michael Rubenstein 4, Gretchen K Hubbard 1, Maria C Moncaliano 1, Andrew Hruszkewycz 5, Ajay Vaghasia 3, Srinivasan Yegnasubramanian 3,6,7, Sarah J Wheelan 3,6,7, Alan K Meeker 1,3,7, Christopher M Heaphy 1,3,7, Mindy K Graham 1,3,7, Angelo M De Marzo 1,3,6,7,*
PMCID: PMC5801764  NIHMSID: NIHMS938282  PMID: 28888037

Abstract

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate-limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high-grade prostatic intraepithelial neoplasia or PIN) and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in eight cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma and that its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target.

Keywords: TERC telomerase RNA component, telomerase, prostate carcinoma, RNA in situ hybridization, MYC

Introduction

Telomeres are unique structures located at the ends of eukaryotic linear chromosomes that protect chromosomes from catastrophic end-to-end fusions and act as a buffer against the loss of terminal DNA that occurs with each round of cell division [1]. To maintain telomere length, some cells express telomerase, a ribonucleoprotein enzymatic complex that extends telomeric DNA (repeated units of TTAGGC) to counterbalance telomere shortening across cell divisions [2]. Telomerase consists of at least two essential elements, a catalytic protein component (TERT) for adding telomeric DNA repeats onto chromosome ends and an RNA subunit (hTR or TERC) that serves as the template for telomere repeat addition [3,4].

In most normal cells, telomerase activity (TA) is undetectable. However, low levels of TA are found in stem and progenitor compartments of tissues that require rapid self-renewal [5,6]. In contrast, most human cancers (80–90%) and immortalized cell lines exhibit increased TA [6,7]. Furthermore, recent studies have uncovered mutations in the promoter region of TERT that result in increased telomerase expression and activity in a number of cancer types such melanoma and bladder carcinoma [8,9].

TA is detectable in the majority of prostate cancers (PCas) [10–14] and its main known precursor lesion, high-grade prostatic intraepithelial neoplasia (HGPIN) [14]. However, the expression levels of the components/subunits of the telomerase holo-enzyme have not been well elucidated at the individual cellular level in situ in PCa tissues. Further, enzyme activity assays alone are unlikely to decipher the intricacies of telomerase regulation, particularly in light of new findings of potential non-telomeric roles in cancer [15,16].

Much of the early research in this area focused on TERT instead of TERC, since TERT mRNA levels correlated better with enzymatic activity across normal tissues and cancer cells. Also, a number of different cell types in culture could be immortalized after forced TERT expression alone [17,18], indicating that TERT is the limiting telomerase component, sufficient for cell immortalization. In addition, TERC was reported to be ubiquitously expressed in different types of normal tissues and its expression did not correlate well with telomerase enzyme activity [19]. Nevertheless, accumulating evidence supports the concept that both components (TERT and TERC) may be altered in cancer [20–22]. For example, in a number of cancer types, including head and neck squamous cell carcinoma, lung squamous cell carcinoma, and cervical carcinoma, there is frequent amplification of the TERC locus [23]. Further, a number of studies have highlighted the importance of TERC in cancer telomere regulation [3,20,24,25], as well as possible TA-independent functions for TERC [15,16,26].

Earlier studies in PCa using radioactive in situ hybridization and quantitative polymerase chain reaction (qPCR) assays have shown that TERC expression may be elevated in PCa tissue [27–30]. However, none of these prior studies provided detailed information about TERC expression in different types of prostatic lesions during PCa development, nor did they perform mechanistic studies to elucidate how TERC expression is regulated during prostate carcinogenesis. Finally, there are inconsistent findings regarding the correlation between TERC expression, TA, Gleason score, and recurrence [10,27,29,30].

MYC is overexpressed in most PCas and HGPIN [31,32], and the MYC locus at chromosome 8q24 is commonly amplified in primary and metastatic PCas [33]. MYC is known to regulate TERT in a number of cell types and hence to drive TA [34–36]. However, the mechanisms regulating TERC expression in PCa are largely unknown.

In this study, we found that TERC is overexpressed consistently in PCa tissues compared with matched normal prostate tissues. Also, we validated a novel branched DNA-based in situ hybridization assay to comprehensively study TERC expression during disease progression in clinical samples from various stages of disease progression. We uncovered the first evidence that TERC expression in cancer is controlled by MYC and determined that TERC depletion by short interfering RNA (siRNA) in PCa cells results in impaired proliferation without increased apoptosis.

Materials and methods

Additional details are provided in the supplementary material, Supplementary materials and methods.

Patients and tissue selection

This study was approved by the Johns Hopkins institutional internal review board. Samples were obtained from 149 patients undergoing radical prostatectomy at Johns Hopkins Hospital (Baltimore, MD, USA). Selection criteria were randomized. Of these, 66 patient tissue samples were used for tissue microarray (TMA) construction and 33 samples were used to create a set of adjacent whole-tissue sections with a considerable number of prostatic intraepithelial neoplasia (PIN) lesions and atrophic regions that are not usually prevalent in TMAs due to limited tissue sampling. All surgical specimens were processed according to institutional standard guidelines. The remaining 50 patient samples consisted of aliquots of RNA prepared from fresh-frozen prostate tissue tumor/benign (T/B) pairs, and used for RNAseq and RT-qPCR. We included additionally castration-resistant prostate cancer (CRPC) tissue samples from eight different rapid autopsies performed in our institution. Tissue collection, preparation, histological categorization, RNA extraction, and quantification were performed following the principles described by the Prostate Cancer Biorepository Network (PCBN) [37].

The median age of the patients was 60 years (range 41–71 years). The Gleason sum mean was 7 (range 6–9). For pathological stage, we used the current American Joint Committee on Cancer (AJCC) TNM staging system for PCa. The most frequent pathological stage was T3AN0MX, and the stages ranged from T2N0MX to T3BN1MX.

Tissue microarrays, whole-tissue sections, and cell blocks

A total of four high-density TMAs were constructed: three from 66 patient tissue samples and one from cell lines, following a protocol previously described by Fedor et al. [38]. For construction of formalin-fixed, paraffin-embedded (FFPE) cell line blocks, cells grown in culture were harvested, fixed in neutral buffered formalin, and processed into FFPE blocks as previously described [39].

Chromogenic in situ hybridization

Chromogenic in situ hybridization (CISH) was performed by using ACD RNAscope® 2.0–BROWN assays with the probes Hs-TERC (Cat No 312361), Hs-TERT (Cat No 605511), Hs-MYC (Cat No 311761) and Mm-Terc (Cat No 425208), all from Advanced Cell Diagnostics Inc, Hayward, CA, USA. All hybridization and incubation steps were performed using the complete HybEZ™ Hybridization System (Advanced Cell Diagnostics Inc) following the manufacturer’s instructions.

Immunohistochemistry

Immunohistochemistry (IHC) was performed using an anti-c-Myc rabbit antibody (Y69) (ab32072, 1:800; Abcam, Cambridge, MA, USA) and the UltraVision™ Quanto Detection System HRP DAB (Thermo Scientific Inc, San José, CA, USA). IHC for cleaved caspase 3 (Asp175) was performed using anti-cleaved caspase 3 (Asp175) rabbit antibody #9661 (Cell Signaling Technology, Inc, Beverly, MA, USA; 1:50, overnight at 4 °C) using PowerVision detection reagents (Leica Biosystems Inc, Buffalo Grove, IL, USA) following the manufacturer’s instructions. PTEN IHC was performed as described previously [40].

Reverse transcription-quantitative PCR (RT-qPCR) analyses

Methods for RNA extraction and complementary (c) DNA synthesis by reverse transcription are presented in the supplementary materials, Supplementary materials and methods.

Quantitative real-time polymerase chain reaction was carried out using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) for TERC, TERT, DKC1, and TBP measurement in cell lines and TERC, TERT, and TBP in T/B RNA pairs from tissue. PCR primer sequences are shown in the supplementary material, Table S1. qPCR conditions were 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s, and a final melting curve step. All PCR reactions were performed using a CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories). Relative normalized target quantity (ΔΔCt) was determined from duplicated standard curves using TBP as a reference transcript for normalization. RT-qPCR experiments were performed at least in triplicate. Additional details are presented in the supplementary materials, Supplementary materials and methods.

RNAseq

Total RNA was quality controlled using the Agilent 2100 Bioanalyzer (Agilent Technologies, Loveland, CO, USA) with all RNA integrity numbers (RINs) greater than 8. RNA samples were processed using the TruSeq Stranded Total RNA sample preparation kit (Illumina, San Diego, CA, USA) using rRNA depletion without initial poly-A RNA selection to generate strand-specific RNAseq libraries, capable of differentiating overlapping sense and antisense transcripts. Barcoded libraries were quality-controlled using the Kappa PCR kit and pooled in equimolar ratios for subsequent cluster generation and sequencing on an Illumina HiSeq 2500 System (Illumina) to yield ~50 million paired-end 100 × 100 bp tags for each sample. Reads were aligned using RSEM and gene expression measures for TERC, MYC, and TERT were extracted as transcripts per million (TPM). The BAM files corresponding to the TERC, MYC, and TERT loci are available at the NCBI BioProject database (PRJNA397328).

Small interfering RNA transfection

Pools composed of four small interfering RNAs (siRNAs) against MYC (SMARTpool ON-TARGETplus MYC siRNA, Cat No L-003282-02-0005; Dharmacon, part of GE Healthcare, Lafayette, CO, USA) and non-targeting scrambled controls (ON-TARGETplus Non-targeting Pool, Cat No D-001810-10-05; Dharmacon) were transfected at a final concentration of 50 nM. Two types of siRNA against TERC were obtained, one designed by Balakumaran et al. [41] (Part No AM16706; Ambion/Life Technologies, Grand Island, NY, USA) and the other purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA; sc-106994). These were used independently and also pooled at equal concentration and transfected at a final concentration of 30 nM. Lipofectamine® RNAiMAX Transfection Reagent (Thermo Scientific Inc) was used as the transfection vehicle. All transfection reactions were supplemented with Opti-MEM® I Reduced Serum Medium (Thermo Scientific Inc). Cells were fed with fresh complete medium every 2 days.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed using 6 μg of rabbit polyclonal IgG c-Myc antibody (N-262) (sc-764; Santa Cruz Biotechnology, Inc) (undiluted, antibody concentration 200 μg/ml) and the iDeal ChIP-seq kit for Transcription Factors (Diagenode, Denville, NJ, USA) following the manufacturer’s instructions, using CCCTC-binding factor (CTCF) and IgG antibodies (provided with the kit) as positive and negative controls, respectively. The efficiency of ChIP of TERC was expressed as the recovery of the different loci calculated as the percentage of the input (relative amount of immunoprecipitated DNA compared with input DNA), using the formula % recovery = 2(Ctinput − Ctsample), where Ctinput and Ctsample are the threshold cycles from the exponential phase of the qPCR for the immunoprecipitated DNA and input, respectively.

Immunoprecipitated DNA was used for qPCR reactions. Primers are listed in the supplementary material, Table S1.

Cell cycle analysis

The incorporation of 5-ethynyl-2′-deoxyuridine (EdU), with propidium iodide (PI) staining, and proliferation analysis of PC-3 cells were assessed after 3 days of TERC knockdown with siRNA. First, cells were pulsed with 10 μM EdU for 2 h, followed by trypsinization and cell counting as previously described. Then, cells were washed with 5 ml of PBS and pelleted by centrifugation at 96 × g. Cell pellets were resuspended in 100 μl of PBS and immediately fixed with 3 ml of ice-cold 100% methanol and acetone (1:1) in a drop-wise manner with intermittent gentle vortexing. Cell permeabilization and Click-iT® reactions were performed by using the Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay according to the manufacturer’s instructions (Thermo Scientific Inc) and samples were counter-stained for DNA content using PI/RNase Staining Buffer (BD Biosciences, San José, CA, USA). Negative controls for each staining (EdU-Alexa488 and PI) were also used to evaluate spectral overlap.

To determine the apoptotic (G0) fraction, PI staining in PC-3 cells was performed after 24, 48, and 72 h of TERC knockdown with siRNA. At each time point, cells were trypsinized and counted. Cells were fixed and washed as described above. DNA content was stained using the PI/RNase staining buffer.

Flow cytometry and cell sorting

Cell populations with MYC overexpression were sorted using an Aria II (BD Biosciences) with Diva software (v. 6.0; BD Biosciences). DNA content experiments were performed on a FACSCalibur (BD Biosciences) and analyzed with FlowJo software (FlowJo v. 10.1r5; FlowJo, LLC, Ashland, OR, USA). A total of 10 000–50 000 events were acquired for analysis.

Luciferase reporter assays

PC-3 cells at different cell densities (1000 or 500 cells per well) were plated in 96-well plates and reverse co-transfected with 2.5 ng of the TERC promoter reporter plasmid (Nluc/TERCProm); 6.25 ng of pGL4.10, which contains luc2 (Photinus pyralis) driven by a CMV promoter, as an internal control (Figure 3A); and a pool of siRNA against MYC, using Lipofectamine® 3000 Reagent (Thermo Scientific Inc) and Opti-MEM® I Reduced Serum Medium (Thermo Scientific Inc). Plates were incubated at 37 °C for 72 h and then enzymatic reactions were carried out by using the Nano-Glo® Dual-Luciferase® Reporter (NanoDLR) Assay (Promega, Madison, WI, USA). Luciferase activity for both constructs was measured using a Wallac Jet 1450 microbeta liquid scintillation and luminescence counter 1450 (Perkin Elmer, Boston, MA, USA). Data were normalized by calculating the ratio of the experimental reporter (Nluc/TERCProm) over the control reporter (luc2/CMV). Values are expressed as the mean with standard deviation (SD) for six replicates.

Figure 3.

Figure 3

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MYC regulation of and binding to TERC promoter. (A) Schematic representation of the human TERC promoter (1.5 kb) + Nluc experimental reporter construct and CMV promoter + luc2 as internal normalization control construct. (B) Promoter activity reduction was observed 72 h after co-transfection of [Nluc + TERCProm], [luc2 + CMV], and MYC siRNA (Wilcoxon rank-sum test). (C) The sizes of the amplified regions are 91 bp of the TERC promoter and 163 bp of the distal gene region and immediate downstream region. (D) ChIP was performed on PC-3 and LNCaP cells using MYC and control antibodies from the iDeal ChIP-seq kit for transcription factors. Sheared chromatin from four million cells, 1 μl of the positive control antibody (CTCF), and 1 μl of the negative IgG control were used per immunoprecipitation. QPCR was performed with PPAT/PAICS and PFAS as positive controls and the non-coding regions of LIN28 and RP11-238I10.1. The recovery is expressed as a percentage of input (the relative amount of immunoprecipitated DNA compared with input DNA after qPCR analysis). At least two independent experiments were performed. Comparison of medians of relative TERC promoter activity between scrambled-siRNA controls and MYC siRNA and percent input between MYC and IgG ChIP in all loci and cell lines was performed by the Wilcoxon rank-sum test. Enriched MYC binding was observed at the promoter and downstream region of TERC. *, **, ***, and **** represent p ≤ 0.05, p < 0.01, p < 0.001, and p ≤ 0.0001, respectively. ns = not significant.

Animal studies

All the samples were obtained from genetically engineered mouse PCa models from a previous study [40]. RNA aliquots from frozen prostate tissue and FFPE tissue were obtained from animal models of isolated MYC overexpression (Hoxb13-MYC mice), targeted Pten loss (Hoxb13-Cre|PtenFl/Fl mice), and mutant mice with Hoxb13-driven overexpression of MYC and with Hoxb13-mediated conditional disruption of Pten alleles (Hoxb13-MYC+|Hoxb13-Cre+|PtenFl/Fl), known as BMPC mice. A full description of the transgenic mice used in this study can be found in Hubbard et al.’s study [40]. Tissues from mice of the FVB/N strain were used as negative controls.

Statistical analysis

Statistical analyses were performed using Stata/SE 14.1 (StataCorp LP, College Station, TX, USA) and GraphPad Prism 6 (GraphPad Software, Inc, La Jolla, CA, USA). Data were tested for normal distributions by using histograms and Kolmogorov–Smirnov and Shapiro–Wilk tests. Differences for RNAseq analysis of TERC and TERT expression in tumor/benign pairs were evaluated by the Wilcoxon signed-rank test. Differences for RT-qPCR analysis of TERC and TERT expression in tumor/benign pairs were calculated by the paired Student’s t-test and Wilcoxon signed-rank test, respectively. Correlation analysis of TERC CISH and TERC expression by RT-qPCR was performed by Spearman’s rank correlation coefficient. Comparisons of TERC CISH nuclear area ratio among normal and other diagnoses were performed by the Wilcoxon rank-sum test (Mann–Whitney). Correlation analysis of the TERC CISH nuclear area ratio and the MYC IHC nuclear area ratio was performed by Pearson’s correlation coefficient. Comparison of medians of TERC expression between scrambled-siRNA controls and MYC-siRNA by RT-qPCR, MYC and TERC levels by RT-qPCR after forced MYC overexpression, relative TERC promoter activity between scrambled-siRNA controls and MYC-siRNA, and percent input between MYC and IgG ChIP in all loci and cell lines was performed using the Wilcoxon rank-sum test. Comparison of TERC expression by RT-qPCR among normal and other diagnoses in the mouse models was performed by the Wilcoxon rank-sum test. Comparison of cell growth curves between scrambled-siRNA controls and TERC and MYC siRNA samples in all cell lines was performed by two-way ANOVA. Comparison of the percentages of cells in cell cycle phases between scrambled-siRNA controls and TERC-siRNA was performed by the paired t-test. Box-and-whisker graphs are bounded by the 25th and 75th percentiles, and whiskers extend to the minimum and maximum data values. For bar graphs, error bars depict the SD. For all comparison graphs, *, **, ***, and **** represent p ≤ 0.05, p < 0.01, p < 0.001, and p ≤ 0.0001, respectively.

Results

Analytical validation of TERC expression using CISH

We evaluated TERC expression in an RNAseq dataset derived from 25 fresh-frozen PCa and matched benign tissues and found that TERC was increased in carcinoma (p < 0.0001, Wilcoxon matched-pairs signed rank test; median fold increase of tumor to normal = 2.2 and mean fold increase = 2.75) (Figure 1A and supplementary material, Table S2). Highly similar results were obtained by RT-qPCR in a separate set of 25 tumor/benign frozen prostate tissue pairs, although the fold difference was greater (p < 0.0001, Wilcoxon matched-pairs signed rank test; median fold increase of tumor to normal = 3.5 and mean fold increase = 3.8) (Figure 1B).

Figure 1.

Figure 1

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TERC is overexpressed in cancer cell lines and clinical prostate tissues. TERC levels are increased in fresh-frozen PCa compared with normal tissue in (A) 25 T/B pairs by RNAseq (Wilcoxon signed-rank test) and (B) 25 T/B pairs by RT-qPCR (Student’s t-test). Box plot boxes are bounded by the 25th and 75th percentiles, with the median also shown. Whiskers extend to the minimum and maximum data values. (C) Chromogenic in situ hybridization (CISH) using RNAScope for TERC in cancer cell lines and normal cells in culture lines (200× original magnification). (D) Scatter plot comparing image analysis data for TERC CISH and qRT-PCR for TERC (Spearman correlation coefficient). (E) Human prostate tissue hybridized for TERC CISH showing strong nuclear signals in PCa (630× original magnification). (F, G) TERC nuclear area ratio determined by image analysis is significantly elevated in the majority of PIN (low-grade, LGPIN and high-grade, HGPIN), PCa, and CRPC samples compared with normal epithelium (Wilcoxon rank-sum test), with representative images shown in H (200× original magnification). *, **, ***, and **** represent p ≤ 0.05, p < 0.01, p < 0.001, and p ≤ 0.0001, respectively.

We next validated a CISH assay to detect TERC in clinical tissue samples. To simulate tissue samples and to establish the specificity of the assay, we performed CISH for TERC on FFPE cell lines. As a negative control, we used U-2 OS cells, which are known to be negative for TA and TERC expression [26,42] and which we verified by RT-qPCR. As expected, no hybridization signals were detected in these U-2 OS cells (Figure 1C and supplementary material, Figure S1A). Conversely, hybridization in cancer cell lines with known TA showed robust signals for TERC within nuclei (Figure 1C). The CISH signals using the TERC probe in the cell lines with known TA were specific for RNA (and not DNA), since they were abolished by pre-hybridization digestion with RNase A (Qiagen, Valencia, CA, USA) (supplementary material, Figure S1B) but not by DNAse (not shown). We also performed CISH on cells that were subjected to TERC knockdown by siRNA, which was verified by RT-qPCR, and confirmed a decrease of TERC hybridization signals (supplementary material, Figure S1C). The knockdown of TERC in cells transfected with siRNA against TERC, as well as the lack of TERC expression in U-2 OS, was verified by RT-qPCR (Figure 1D). Taken together, these findings establish the specificity of the TERC CISH assay.

To more fully determine the quantitative nature of the TERC CISH assay, we quantified TERC CISH in 11 cell lines/primary cultures subjected to FFPE treatment by computerized image analysis (the methods used for quantitative image analyses can be found in the supplementary material, Supplementary materials and methods) and compared the results with RT-qPCR expression for TERC in these same cell lines. Figure 1D shows that there was a significant positive correlation between TERC levels determined by CISH and RT-qPCR (Spearman rank correlation coefficient = 0.84, p = 0.0013). CISH in benign/non-immortalized prostatic epithelial (PrECs) or stromal cells (PrSCs) in culture had low amounts of TERC by both CISH and RT-qPCR, but the levels were lower than in the prostate or other cancer cell lines except U-2 OS, in which TERC expression was absent by both methods.

CISH shows that TERC is overexpressed in PCa and PIN

We next examined TERC expression using CISH in different prostatic lesions using standard whole slides and TMAs. In benign glands and atrophy, the overall staining was low, with only sparse numbers of relatively weak intensity (usually less than five) nucleoplasmic dots. Interestingly, the majority of HGPIN and carcinoma lesions showed elevated expression of TERC compared with normal-appearing epithelium in the same patient samples, and the signals appeared to be primarily nucleolar and nucleoplasmic (Figure 1E, H). In terms of tumor grade, TERC expression was high in all Gleason patterns, with no increasing or decreasing trends in levels among them (supplementary material, Figure S2A). In terms of tumor stage, there were also no specific trends in TERC levels in these clinically localized disease samples (supplementary material, Figure S2B). TERC was also highly expressed in all the CRPC samples obtained from eight autopsies and analyzed by qualitative visual scoring (Figure 1H and supplementary material, Figure S3). Thus, TERC overexpression appears to occur early in prostate carcinogenesis and persists at all stages of disease progression examined, including lethal metastatic disease.

Examination of TERC expression in online publically available databases including cBioPortal (http://www.cbioportal.org/) [43,44] and Oncomine™ (http://www.oncomine.com, December 2015; Thermo Fisher Scientific, Ann Arbor, MI, USA) revealed a single study in PCa in which both tumor and benign samples were reported that had data on TERC RNA levels, and this dataset shows a clear increase in carcinoma compared with normal/benign (supplementary material, Figure S4). We also examined TERC gene alterations and expression using different cancer studies found in the same databases and found that, as previously reported, the TERC locus was commonly amplified in lung, ovarian, esophagus, cervical, and head and neck carcinomas, especially of squamous histologic type (supplementary material, Figure S5A). In PCa datasets, TERC amplification ranged from 0 to 7.5% (supplementary material, Figure S5B) and there appeared to be a weak correlation between TERC expression and gene copy number (supplementary material, Figure S5B–D). Interestingly, no mutations in TERC have been identified in human cancer to date.

TERT mRNA is known to be upregulated in PCa. To verify this in our samples and to determine whether TERT mRNA levels correlate with TERC RNA, we performed RT-qPCR for TERT mRNA and this was increased in carcinoma as expected (supplementary material, Figure S6A, B); there was a statistically significant moderate positive correlation between TERC and TERT RNA expression in all tissue samples by RNAseq and qRT-PCR (supplementary material, Figure S6C, D). The levels of TERT mRNA (while not strictly informative in terms of TERT protein) were much lower than those of TERC in both the RT-qPCR and the RNAseq datasets, which is consistent with the very low levels of TERT mRNA seen previously by many others. We then performed RNA CISH for TERT and found it to be readily detectable in cells transfected with an expression vector encoding human TERT cDNA or in cells infected with a lentiviral construct containing human TERT (supplementary material, Figure S7A) but undetectable in negative control cells including wild-type mouse embryo fibroblasts (MEFs) or MEFs from mice with biallelic disruption of Tert (supplementary material Figure S7B). Also, we could detect low levels of CISH signals for TERT in FFPE telomerase-positive PCa cells processed into FFPE pellets (supplementary material, Figure S7C), but we could not detect any signals in PCa whole-tissue sections (not shown), consistent with our findings of very low TERT mRNA levels in fresh-frozen PCa specimens. Antibodies for TERT protein are not useful at this time for IHC [45,46].

MYC regulates TERC expression in cancer cell lines

MYC protein levels are elevated in human HGPIN and most PCa specimens [31], which is similar to the current findings for TERC. Taken together with the fact that MYC is known to regulate TERT and other telomerase components, we therefore examined whether there was a correlation between the levels of MYC protein and TERC in the clinical PCa samples using both adjacent whole-tissue sections and TMAs analyzed for TERC with CISH and MYC by IHC (Figure 2A and supplementary material, Figure S8). By quantitative image analysis, there was a positive correlation between TERC CISH and MYC IHC in both TMAs and whole-tissue sections (Pearson correlation coefficient = 0.54, p < 0.001, and 0.51, p < 0.001, respectively) (supplementary material, Figure S8A, B). Positive correlations between MYC and TERC were found in atrophy, HGPIN, and carcinoma (supplementary material, Figure S8D–F). We also found a positive correlation by querying our RNAseq data (supplementary material, Figure S9A) and using a publically available PCa study [47] (supplementary material, Figure S9B).

Figure 2.

Figure 2

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MYC regulates TERC expression. (A) TERC expression in situ correlates with MYC protein in TMAs (×200 original magnification) and whole-tissue sections. (B) Decrease in TERC expression at days 2 and 6 after MYC knockdown. Data were log2 transformed and error bars indicate the SD of at least three experimental replicates. Comparison of medians of expression was performed by the Wilcoxon rank-sum test. (C) Induction of TERC following forced MYC overexpression in DU-145, LNCaP, and PC-3 (Wilcoxon rank-sum test). (D) Mouse Terc is upregulated in mice overexpressing human MYC in the mouse prostate (Hoxb13-MYC) and in mice overexpressing MYC with combined knockout of Pten (BMPC) by RT-qPCR in fresh-frozen prostate samples (Wilcoxon rank-sum test) and (E) confirmed by CISH and IHC staining in FFPE tissue (×200 original magnification). *, **, ***, and **** represent p ≤ 0.05, p < 0.01, p < 0.001, and p ≤ 0.0001, respectively. ns = not significant.

We next sought to determine whether MYC regulates TERC levels. For this, we knocked down MYC with siRNA in five human PCa cell lines (CWR22rv1, DU-145 L, LAPC-4, LNCaP, and PC-3), one lung non-small cell carcinoma cell line (NCI-H23), one breast carcinoma cell line (MCF-7), and one colorectal carcinoma cell line (DLD-1) (supplementary material, Figure S10). There was an overall and progressive decrease in TERC expression as measured by RT-qPCR, which was especially pronounced after 6 days of transfection in all cell lines (Figure 2B). The levels of TERT and DKC1 mRNA were also determined (supplementary material, Figure S11), since these genes have been shown to be regulated by MYC [34–36,48]. There were decreases in both TERT and DKC1 (the latter evaluated only in PC-3 and LNCaP) expression after MYC knockdown. Interestingly, TERT expression showed an upward trend in PC-3 at day 6. Using lentiviral vectors, we forced overexpression of MYC in LNCaP, DU-145, and PC-3 cell lines (supplementary material, Figure S12). As shown in Figure 2C, cells overexpressing MYC consistently had higher levels of TERC when compared with empty control vector.

To determine whether human MYC regulation of TERC is conserved across species, we examined the expression of mouse Terc after forced overexpression of MYC conditionally in the mouse prostate (Hoxb13-MYC mice), or by a combination of overexpression of MYC with prostate-specific Pten deletion (BMPC mice) [40]. Interestingly, using RT-qPCR to measure mouse Terc, PIN samples from the Hoxb13-MYC model showed a marked increase in Terc expression compared with normal mouse prostate (FVB/N) (Figure 2D), indicating that human MYC overexpression in the mouse prostate can induce mouse Terc expression in vivo. In BMPC mice, we found Terc overexpression in PIN, invasive adenocarcinoma, and metastases compared with normal prostate tissues from wild-type mice as measured by RT-qPCR (Figure 2D). Mouse Terc overexpression in response to MYC overexpression was confirmed by CISH for mouse Terc in all of these mouse lesions (Figure 2E and supplementary material, Figure S13). Interestingly, deletion of Pten alone, also associated with development of PIN in the mouse prostate, did not result in mouse Terc overexpression (Figure 2D, E), indicating specificity of Terc/TERC induction by MYC and not simply as a secondary event associated with carcinogenesis.

To explore whether the regulation of TERC by MYC was occurring at the transcriptional level, we cloned the human TERC promotor region [1.5 kb upstream of the TERC transcription start site (TSS)] into a luciferase reporter (Figure 3A). After co-transfection of PC-3 cells with MYC siRNA, we observed reduced promoter activity (Figure 3B), although MYC knockdown did not suppress the activity of the control vector (pGL4.10[luc2/CMV]) (supplementary material, Figure S14). We performed chromatin immunoprecipitation (ChIP) in LNCaP and PC-3 cells and found MYC enrichment at both the TERC promoter and the terminal region in both cell lines (Figure 3D). We also examined the UCSC Genome Browser database including data from two different human genome assemblies and the ENCODE annotation database [49] and observed peaks of MYC occupancy mainly upstream of the TERC TSS and less towards the end of the gene and immediately following downstream region in a number of other cell types (supplementary material, Figure S15). Taken together, these findings suggest that the induction of TERC expression in response to MYC is most likely due to direct cis activation of TERC transcription. While there are no canonical E-boxes in a range of 1 kb upstream and 1 kb downstream the TERC TSS, there are three non-canonical E-boxes (CAGCTT at −61 bp, CACATG at +179 bp, and CAGCTG at +444 bp; supplementary material, Figure S16), with the upstream and downstream non-canonical E-boxes overlapping with the regions of MYC enrichment in our ChIP analyses.

To determine whether forced reductions in MYC result in decreased TA, we measured TA in LNCaP and PC-3 cells after 48 h of siRNA-mediated MYC knockdown. As expected, MYC-depleted cells showed a reduction in TA (supplementary material, Figure S17).

TERC is required for proliferation of PCa cells

Prior work has demonstrated that forced reductions in endogenous levels of TERC inhibit growth and induce apoptosis in lymphoma, melanoma, bladder, breast, and colorectal carcinoma cell lines [15,16,26]. We performed knockdown experiments for TERC in PCa cell lines, with two different siRNAs complementary to the template sequence and the pseudoknot core domain, separately. Since knockdown efficiency was improved when combining both siRNAs, we pooled them for transfection in LNCaP and PC-3 cells (supplementary material, Figure S18). We also performed MYC knockdown using siRNA in LNCaP and PC-3 cells as positive controls for reduced cell proliferation.

TERC knockdown caused a growth delay in both cell lines in this transient knockdown experiment, with a reduction in growth apparent in the initial setting of knockdown, followed by resumption of growth between days 4 and 6 (Figure 4A, B). We analyzed the cell cycle distribution of PC-3 cells after forced reductions in TERC by siRNA. These results are shown in Figure 4C, which illustrates a representative 2D cell cycle plot, and Figure 4D, which shows the summary of three independently performed experiments from the third day results. Statistical analysis indicates a significant increase in the S-phase fraction (paired t-test, p = 0.002 at 3 days and p = 0.03 at 6 days). These results suggest an S-phase arrest/accumulation in PC-3 cells subjected to forced reductions in TERC. Also consistent with such a block, there were fewer cells in G2/M (Figure 4C, D). TERC knockdown did not induce apoptosis in PC-3, as seen by a lack of a sub-G0/G1 population in TERC knockdown (Figure 4E and supplementary material, Figure S19) and cells stained with cleaved caspase 3 IHC (Figure 4F). Similar results in terms of growth inhibition, albeit not as pronounced, were obtained when each of the anti-TERC siRNAs were used separately (supplementary material, Figure S20). Along with reductions in TERC levels, siRNA-mediated TERC knockdown resulted in a decrease in TA in LNCaP and PC-3 cells (supplementary material, Figure S21). This indicates that despite the findings that TERC levels appear to be much higher than needed to match TERT protein levels stoichiometrically [21], forced reductions in TERC caused a corresponding reduction in TA, indicating that TERC levels may also be rate-limiting for telomerase activity in PCa cells.

Figure 4.

Figure 4

Open in a new tab

TERC is required for PCa cell proliferation. (A, B) Cell growth inhibition induced by siRNA-mediated TERC depletion in LNCaP and PC-3 cells. (C) Representative plots of cell cycle analysis with EdU + propidium iodide staining, followed by flow cytometry in PC-3 cells, showing arrest in S-phase after TERC siRNA transfection and (D) summary of three independently performed experiments at day 3 (paired t-test). (E) TERC knockdown does not induce apoptosis in PC-3 when measured by quantification of sub-G1 population and (F) cleaved caspase 3-positive cells. *, **, ***, and **** represent p ≤ 0.05, p < 0.01, p < 0.001, and p ≤ 0.0001, respectively. ns = not significant.

Discussion

In this study, we show by multiple cross-validated approaches, including an in situ approach, that TERC is overexpressed in all phases of prostate carcinogenesis. We also show that TERC expression is regulated by MYC in prostate cancer: (i) TERC and MYC expression levels were correlated across normal prostate glands, precursor lesions, and carcinoma; (ii) forced reduction of MYC resulted in decreased TERC levels in eight cancer cell lines (prostate, lung, breast, and colorectal); (iii) forced overexpression of MYC in PCa cell lines and in the mouse prostate resulted in increased TERC levels; (iv) there was a decrease in human TERC promoter activity with MYC silencing; and (v) MYC occupies the TERC locus, as assessed using ChIP in a number of cell lines. In terms of the functional consequence of alterations in TERC overexpression, we found that forced reductions of TERC in a number of PCa cell lines resulted in decreased cell proliferation, supporting TERC as a potential target for therapeutic intervention in prostate cancer [16].

TERC overexpression in cancer may be partly due to recurrent amplification and copy number gains [50–53]. In PCa, publically available data show a range of TERC gene amplification in PCa from 0–4.2% in primary untreated tumors to 7.4% in CRPC. This provides additional support suggesting an oncogenic role for TERC in PCa, although it certainly does not explain the much more frequent overexpression that we report here; the present findings suggest that MYC overexpression may be largely responsible. Additional studies are required to determine the relationship between the expression levels of TERC RNA and TERC gene amplification in prostate and other cancers.

MYC has been shown to regulate TA in a number of prior studies [34,54,55]. Since then, several reports have identified that a number of components of the telomerase holoenzyme, including TERT, DKC1, and NHP2L1, by transcriptional regulation experiments [35,36,48,56–60], and WDR79 (WRAP53), RUVBL1 (PONTIN), RUVBL2 (REPTIN), and NOLA1, by expression array data [61], may be regulated by MYC. Further, while the precise molecular mechanisms by which MYC regulates the TERT promoter remain unclear, it is possible that TERT promoter mutations, which are found in a number of cancer types (e.g. bladder, melanoma, etc), may ultimately promote increased MYC binding to cis-E-boxes as a result of increased ETS/TCF binding that can cooperate with MYC [62]. It has also been demonstrated that TERT reactivation is able to regulate MYC-dependent oncogenesis in a manner that is independent of telomerase activity [63]. To our knowledge, no prior studies have shown that MYC regulates TERC. Interestingly, MYC has been shown to regulate a viral telomerase RNA gene (vTERC) in Marek’s disease virus (MDV)-transformed lymphoblastoid cell line MSB-1 [64–66]. Shkreli et al. demonstrated that the transcriptional activity of the vTERC promoter resulted largely from an E-box, and MYC binding was demonstrated by ChIP assays [64]. We also found in the present study that forced reductions in MYC resulted in reduced levels of DKC1 in LnCAP and PC-3 cells. Since DKC1 protein can stabilize TERC RNA [67], it is possible that the reduced TERC levels after MYC knockdown and the increased TERC levels after MYC overexpression may also be related in part to DKC1-mediated TERC stabilization. Additional studies are required to determine the overall contribution of direct transcriptional activation by MYC and MYC-mediated DKC1 stabilization of TERC.

Using a combination of two different siRNAs, we found that TERC knockdown elicits significant growth inhibition in PCa cell lines. In the past, several studies have targeted TERC with RNAi [15,16,26,68], which has consistently shown a reduced growth rate in different types of cancer cell lines [15,16,68–71] and inhibited xenograft tumor growth in mice [68,72]. The inhibitory effect on cell proliferation that we noticed was modest, which is in keeping with previous reports [16,26,68,70,72] including one with PCa cell line [73]. Although some studies have shown that cell growth inhibition in TERC-depleted or TERC-template-mutant cancer cells may be due to telomerase-independent functions [15,16], whether the reduced proliferation found in our study is strictly related to a reduction in TA or whether it is at least in part related to a telomerase-independent mechanism is unclear. While the mechanisms by which TERC knockdown could affect cell cycle arrest were not elucidated in our study, Kedde et al [26] found that TERC can restrain ATR activity, and thus knockdown of TERC resulted in induction of an ATR-mediated cell cycle arrest, albeit in G2/M and not S-phase. The results from Kedde et al were partially dependent on p53, although ours were not because PC-3 cells are TP53-null. Further, Li et al reported a TP53-independent role for TERC in effecting cell cycle arrest as well (also in G2/M arrest), which could relate to effects of TERC on cyclin G2 [74]. Several prior studies have shown an increase in apoptosis in response to forced reductions of TERC, although the percentage of the apoptotic fraction varied across different types of cell lines [15,68,70,75–77]. We found no change in the apoptotic fraction. It is possible that this may depend on the specific cell types analyzed or on the different experimental designs.

In summary, our current study shows that TERC is consistently overexpressed in PIN and PCa. We also show the first evidence of MYC regulation of TERC, suggesting the novel hypothesis that MYC may regulate virtually all telomerase components. Additionally, we show that growth inhibition and no significant alteration in apoptosis occur after siRNA-mediated TERC knockdown in PCa cells. Our results contribute to observations suggesting that TERC plays an important role in carcinogenesis and highlights the potential importance of inhibitors of both main telomerase components [78] as a therapeutic approach in telomerase-positive cancers.

Supplementary Material

Suppl FigS1. Figure S1.

Quality control of TERC CISH staining

Suppl FigS10. Figure S10.

Efficacy of siRNA-mediated MYC knockdown in cancer cell lines as verified by western blotting

Suppl FigS11. Figure S11.

MYC knockdown decreases TERT and DKC1 levels in cell lines

Suppl FigS12. Figure S12.

Forced overexpression of MYC results in increased TERC levels in cancer cell lines

Suppl FigS13. Figure S13.

MYC causes overexpression of mouse Terc in the prostates of mice overexpressing human MYC

Suppl FigS14. Figure S14.

Promoter reporter activity of the control vector

Suppl FigS15. Figure S15.

MYC binding to TERC locus in different cell lines (MCF-7, HUVEC, HeLa, HepG2, K562, H1-hESC, and GM12878)

Suppl FigS16. Figure S16.

Location of potential MYC binding sites (e-boxes) in the TERC locus

Suppl FigS17. Figure S17.

Reduction in telomerase activity after MYC knockdown

Suppl FigS18. Figure S18.

TERC knockdown efficacy, using a pool of siRNAs, was confirmed by RT-qPCR

Suppl FigS19. Figure S19.

DNA content of PC-3 cells transfected with scrambled non-targeting siRNA and TERC siRNA pool, stained with propidium iodide and analyzed by flow cytometry

Suppl FigS2. Figure S2.

Correlation of TERC levels with Gleason grade and stage

Suppl FigS20. Figure S20.

Cell growth inhibition induced by siRNA-mediated TERC depletion in LNCaP and PC-3 cells

Suppl FigS3. Figure S3.

TERC overexpression in metastatic castrate-resistant prostate cancer (CRPC) samples

Suppl FigS4. Figure S4.

TERC gene expression in a publically available online dataset

Suppl FigS5. Figure S5.

TERC gene amplification in publically available online datasets

Suppl FigS6. Figure S6.

TERT expression and relation to TERC

Suppl FigS7. Figure S7.

RNA CISH for TERT in FFPE cell blocks

Suppl FigS8. Figure S8.

Positive correlation between TERC CISH and MYC IHC in different prostate diagnoses

Suppl FigS9. Figure S9.

Correlation of TERC and MYC levels

Suppl Legends
Suppl Materials
Suppl tableS2. Table S2.

TPM values from RNAseq for MYC, TERC, and TERT, as well as clinico-pathological variables of specimens used for RNAseq

Suyppl FigS21. Figure S21.

Reduction in telomerase activity after TERC knockdown

suppl table S1. Table S1.

Primers used for RT-PCR, ChIP, and Gibson assembly

Acknowledgments

This study was supported by the NIH/NCI SPORE in Prostate Cancer: P50CA58236, and the NIH/NCI U01 CA196390, the US Department of Defense Prostate Cancer Research Program (PCRP), DoD Award W81XWH-14-2-0182, the Prostate Cancer Bioreposi-tory Network (PCBN), the Prostate Cancer Foundation, and the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center Experimental and Computational Genomics Core (ECGC) supported by NIH/NCI grant P30 CA006973. We thank ECGC members Anuj Gupta, Alyza Skaist, Paul Schaughency, Jennifer Meyers, and Michael Rongione for their technical support of RNA-seq data collection and processing. We also want to thank Helen Fedor, Marcela Southerland, and Kristen Lecksell for TMA construction and whole slide scanning. We thank Jonathan Coulter, Stefan Barfeld, and Michael Haffner for their valuable insights and help with experimental procedures.

Footnotes

No conflicts of interest were declared.

Author contributions statement

JB, AD, QZ, DE, MR, GH, and MG conceived and carried out experiments. MM carried out experiments. AH, AV, SY, SW, AM, CH, and AD analyzed data. All authors were involved in writing the paper and had final approval of the submitted and published versions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Suppl FigS1. Figure S1.

Quality control of TERC CISH staining

NIHMS938282-supplement-Suppl_FigS1.pdf (1.3MB, pdf)
Suppl FigS10. Figure S10.

Efficacy of siRNA-mediated MYC knockdown in cancer cell lines as verified by western blotting

NIHMS938282-supplement-Suppl_FigS10.pdf (182.1KB, pdf)
Suppl FigS11. Figure S11.

MYC knockdown decreases TERT and DKC1 levels in cell lines

NIHMS938282-supplement-Suppl_FigS11.pdf (227.4KB, pdf)
Suppl FigS12. Figure S12.

Forced overexpression of MYC results in increased TERC levels in cancer cell lines

NIHMS938282-supplement-Suppl_FigS12.pdf (295.6KB, pdf)
Suppl FigS13. Figure S13.

MYC causes overexpression of mouse Terc in the prostates of mice overexpressing human MYC

NIHMS938282-supplement-Suppl_FigS13.pdf (4.9MB, pdf)
Suppl FigS14. Figure S14.

Promoter reporter activity of the control vector

NIHMS938282-supplement-Suppl_FigS14.pdf (76.2KB, pdf)
Suppl FigS15. Figure S15.

MYC binding to TERC locus in different cell lines (MCF-7, HUVEC, HeLa, HepG2, K562, H1-hESC, and GM12878)

NIHMS938282-supplement-Suppl_FigS15.pdf (100.7KB, pdf)
Suppl FigS16. Figure S16.

Location of potential MYC binding sites (e-boxes) in the TERC locus

NIHMS938282-supplement-Suppl_FigS16.pdf (113.5KB, pdf)
Suppl FigS17. Figure S17.

Reduction in telomerase activity after MYC knockdown

NIHMS938282-supplement-Suppl_FigS17.pdf (100.7KB, pdf)
Suppl FigS18. Figure S18.

TERC knockdown efficacy, using a pool of siRNAs, was confirmed by RT-qPCR

NIHMS938282-supplement-Suppl_FigS18.pdf (113.5KB, pdf)
Suppl FigS19. Figure S19.

DNA content of PC-3 cells transfected with scrambled non-targeting siRNA and TERC siRNA pool, stained with propidium iodide and analyzed by flow cytometry

NIHMS938282-supplement-Suppl_FigS19.pdf (192.8KB, pdf)
Suppl FigS2. Figure S2.

Correlation of TERC levels with Gleason grade and stage

NIHMS938282-supplement-Suppl_FigS2.pdf (236.8KB, pdf)
Suppl FigS20. Figure S20.

Cell growth inhibition induced by siRNA-mediated TERC depletion in LNCaP and PC-3 cells

NIHMS938282-supplement-Suppl_FigS20.pdf (387.7KB, pdf)
Suppl FigS3. Figure S3.

TERC overexpression in metastatic castrate-resistant prostate cancer (CRPC) samples

NIHMS938282-supplement-Suppl_FigS3.pdf (456.8KB, pdf)
Suppl FigS4. Figure S4.

TERC gene expression in a publically available online dataset

NIHMS938282-supplement-Suppl_FigS4.pdf (64.7KB, pdf)
Suppl FigS5. Figure S5.

TERC gene amplification in publically available online datasets

NIHMS938282-supplement-Suppl_FigS5.pdf (385KB, pdf)
Suppl FigS6. Figure S6.

TERT expression and relation to TERC

NIHMS938282-supplement-Suppl_FigS6.pdf (398.8KB, pdf)
Suppl FigS7. Figure S7.

RNA CISH for TERT in FFPE cell blocks

NIHMS938282-supplement-Suppl_FigS7.pdf (257.1KB, pdf)
Suppl FigS8. Figure S8.

Positive correlation between TERC CISH and MYC IHC in different prostate diagnoses

NIHMS938282-supplement-Suppl_FigS8.pdf (273.7KB, pdf)
Suppl FigS9. Figure S9.

Correlation of TERC and MYC levels

NIHMS938282-supplement-Suppl_FigS9.pdf (97.8KB, pdf)
Suppl Legends
NIHMS938282-supplement-Suppl_Legends.docx (30.4KB, docx)
Suppl Materials
NIHMS938282-supplement-Suppl_Materials.docx (30.4KB, docx)
Suppl tableS2. Table S2.

TPM values from RNAseq for MYC, TERC, and TERT, as well as clinico-pathological variables of specimens used for RNAseq

NIHMS938282-supplement-Suppl_tableS2.xlsx (11.1KB, xlsx)
Suyppl FigS21. Figure S21.

Reduction in telomerase activity after TERC knockdown

NIHMS938282-supplement-Suyppl_FigS21.pdf (152.3KB, pdf)
suppl table S1. Table S1.

Primers used for RT-PCR, ChIP, and Gibson assembly

NIHMS938282-supplement-suppl_table_S1.xlsx (12.3KB, xlsx)

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