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The transcription factor ERG increases expression of neurotransmitter receptors on prostate cancer cells
© Kissick et al. 2015
Received: 8 January 2015
Accepted: 19 August 2015
Published: 27 August 2015
The TMPRSS2-ERG gene fusion occurs in about half of prostate cancer (PCa) cases and results in overexpression of the transcription factor ERG. Overexpression of ERG has many effects on cellular function. However, how these changes enhance cell growth and promote tumor development is unclear.
To investigate the role of ERG, LNCaP and PC3 cells were transfected with ERG and gene expression and metabolic profile were analyzed.
Our data show that expression of ERG induces overexpression of many nicotinicacetylcholine receptors (nAChRs). In addition, metabolic profiling by LC-MS/MS revealed elevated production of several neurotransmitters in cells expressing ERG. Consistently, treatment of ERG-expressing cells with nicotine induced elevated calcium influx, GSK3β (Ser9) phosphorylation and cell proliferation. Finally, we show that PCa patientswho are smokers have larger tumors if their tumors are TMPRSS2-ERG gene fusion positive.
Collectively, our data suggest that ERG sensitizes prostate tumor cells to neurotransmitter receptor agonists like nicotine.
Transcription factor expression and function is commonly disrupted in cancer, leading to the gain or loss of critical characteristics required for tumor development. One such example, found in 50 % of prostate cancers, involves the fusion between the ETS transcription factor ERG with the androgen-regulated gene TMPRSS2, resulting in overexpression of ERG in the prostate epithelium . Overexpression of ERG drives a number of oncogenic effects in prostate cells. For example, ERG co-operates with the PI3K pathway to promote oncogenesis [2, 3]. ERG also increases androgen receptor (AR) binding in mouse prostate, and increases AR transcriptional output in PTEN-deficient prostate cancer patients . ERG-dependent chromatin modification has also been found by many Chip-Seq studies that also revealed that AR binding is significantly altered in ERG-expressing cells [4–6]. ERG may regulate these changes by altering expression of the histone deacetylase, HDAC1, as this is one of the most up-regulated genes in ERG-positive prostate cancer. ERG also induces expression of EZH2, a H3K27 methyl-transferase critically involved in chromatin modification . Additionally, ERG has been found to enhance signaling through the WNT pathway, an event that results in increased activation of β-catenin [8–10]. While these roles suggest an important role for ERG in tumorigenesis, in vivo mouse models have found that ERG overexpression alone is insufficient to induce cancer [3, 4]. However, patients with ERG-positive high-grade prostatic intraepithelial neoplasia (HGPIN) are much more likely to progress to prostate cancer . Therefore, the question still remains, if the TMPRSS2:ERG fusion alone is not sufficient to cause cancer, what changes does it induce in prostate epithelial cells to predispose them to become cancerous.
Here we investigated the role of ERG in prostate cancer and found that overexpression of ERG resulted in expression of a number of neurotransmitter receptors. We found that these receptors were functional and treatment of ERG-positive cells with the acetylcholine receptor agonist, nicotine, increased cell growth, calcium signaling and GSK-3β (Ser9) phosphorylation. As a result of this, we measured the polar metabolic profile of ERG-positive cells and found several elevated neurotransmitters. Finally, we found that if patients with TMPRSS2:ERG fusion positive prostate cancer were smokers, they had significantly higher number of biopsy cores positive for cancer, and each core contained a larger proportion of tumor tissue, compared to TMPRSS2:ERG negative smokers, suggesting an increased tumor volume in these patients. Together, these data suggest a novel mechanism by which ERG may contribute to prostate tumorigenesis.
Materials and Methods
Patient data & Ethics Statement
Data analyzed in this work was extracted from the BIDMC Early Detection Research Network (EDRN), which enrolled men at 6 clinical practice sites at Harvard, Michigan, and Cornell universities who had undergone their first prostate biopsy were identified. Demographic and pre-biopsy clinical data, including PSA history, digital rectal exam (DRE) results, smoking history, prostate volume and all biopsy procedure details were ascertained on case report forms. Prostate biopsy results were reviewed with reports confirmed by institutional pathologists. Pathology reports included confirmed histology, number of cores and percent of each core involved with carcinoma, and primary and secondary Gleason patterns . Among patients with newly diagnosed (untreated) prostate cancer, TMPRSS2:ERG status was determined using a PCR-based urine TMPRSS2:ERG fusion assay (performed by Gen-Probe, CA) as previously described .
All participants had provided written informed consent and all prostate biopsies had been offered according to National Comprehensive Cancer Network (NCCN) guidelines .
All research carried out on humans was in compliance with the Helsinki Declaration and approved by the Dana-Farber/Harvard cancer Center Internal Review Board protocol #DF/HCC 05-209.
Transfection of cells
ERG-RFP or RFP expression was induced in the PC3 and LNCaP cells using a lentiviral transduction system provided by Dr. Owen Witte (UCLA, Los Angeles, CA) as described previously .
Microarray and PCR gene expression analysis
Gene expression was assessed using Affymetrix’s (Santa Clara, CA) GeneChip Human Genome U133 2.0 arrays. Arrays were scanned with the GeneChip Scanner 3000 and GeneChip Operating Software acquired raw fluorescence signal. Scanned array images were analyzed by dChip  and CEL file data were normalized using RMAexpress (http://rmaexpress.bmbolstad.com)  and annotated using Multiexperiment Viewer software (http://www.tm4.org/mev.html). The data generated have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE55944 (http://www.ncbi.nlm.nih.gov/gds/?term=GSE55944). Primers for select gene subsets were designed using the Primer3 platform (http://bioinfo.ut.ee/primer3/) and analyzed using a StepOnePlus real-time PCR machine from Applied Biosystems (Life Technologies, Grand Island, NY).
Pathway and Upstream Regulator Analysis
Pathway analysis of the differentially expressed RT-PCR-validated genes (Fig. 2) in ERG- and ERG+ LNCap cells was performed using the commercial systems biology oriented package, Ingenuity Pathways Analysis (www.ingenuity.com). The Score (-log p-value) is calculated using Fisher's Exact Test and indicates the likelihood a gene will be found in a network due to random chance. Ingenuity's Upstream Regulator Analysis tool was used to identify the cascade of upstream transcriptional regulators that can explain the observed gene expression changes.
Metabolite content measurement in LNCap-ERG+ and LNCap-ERG- cells and data analysis was performed as described previously [18–20]. Briefly, 106 cells exponentially growing in media were harvested in 3 mL of cold 80 % v/v HPLC grade methanol. Fresh medium was added 24 and 2 h prior to the extraction. Samples were re-suspended in 20 μL HPLC-grade water for mass spectrometry analysis. 10 μL were injected and analyzed using a 5500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX) coupled to a Prominence UFLC HPLC system (Shimadzu) via selected reaction monitoring (SRM) of a total of 256unique endogenous polar metabolites for steady-state analyses. Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v2.0 software (AB/SCIEX). Measurements were performed in triplicate and only metabolites that were determined in all 6 samples were retained, normalized and analyzed using MetaboAnalyst2.0 software (http://www.metaboanalyst.ca/MetaboAnalyst/).
To investigate calcium signaling in prostate cancer cell lines, cells were stained with 1 μM of Fluo-4 AM for 15 min at 37 °C in Hanks Buffered Salt Solution. Cells were then treated with 10 μM solution of nicotine and intracellular Ca2+ concentration over time analyzed by flow cytometry (BeckmanCoulter, Galios).
Pathscan and Western blot analysis of phospho-proteins
Nicotine- or buffer-treated cells were lysed on ice at the various time points with Cell Signaling Technologies cell lysis buffer. Protein concentration was determined using the Thermo Scientific BCA protein assay kit. Analysis of phospho-proteins was performed using Cell Signaling PathScan Akt signaling array (Cat: 9700) as described by the manufacturer’s instructions. Confirmation of PathScan results was performed by western blot. Briefly, protein was separated usingLife Technologies NovexSDS-Page system. Antibodies from Cell SignallingT echnolgy against phospho-GSK3a/b (Ser21/9) (clone 37 F11), β-actin (clone 13E5) and phospho-AKT (Ser129) (clone D4P7F) were used to detect relevant proteins under conditions suggested by the manufacturer.
Statistical analysis was performed using the Student’s T-test. P values of less than 0.05 were considered significant.
ERG overexpression alone induces expression of neurotransmitter receptor genes in LNCaP cells
ERG expression significantly alters the metabolic profile in LNCap cells
Nicotine enhances growth of ERG-positive prostate cancer cells and induces Ca2+ influx and phosphorylation of GSK3β
TMPRSS2:ERG+ prostate cancer patients that smoke have a larger proportion of biopsy core involvement
Given our findings indicating that ERG-positive prostate tumor cells exhibited expression of neurotransmitter receptors and that nicotine enhanced tumor cell growth in vitro, we hypothesized that growth of ERG-positive prostate cancer would be enhanced in patients that smoked. We used data collected from a cohort of patients enrolled through the Early Detection Research Network (EDRN) to correlate biopsy core involvement to ERG and smoking status. Among patients with newly diagnosed (untreated) prostate cancer in this cohort, 62 of these patients had their TMPRSS2:ERG status determined using a urine TMPRSS2:ERG fusion assay (See Additional file 1: Data #1 for all patient data). Of these 62 patients tested, 24 reported being regular smokers and 39 were positive for ERG (Fig. 5a). Interestingly, smokers whose tumors had the TMPRSS2:ERG fusion had a significantly higher number of both biopsy core involvement and total core involvement, compared to patients whose tumors are fusion negative (Fig. 5b and c). Similarly, given a patient was positive for ERG, if they also smoked they had a near significant (0 = 0.055) but no difference in number of cores involved. In contrast, non-smokers had no significant difference in either of these measurements between ERG- and ERG+ cancers. No significant difference in Gleason score or T staging was observed between the ERG groups. These data show in vivo evidence supporting genomic data that suggested that ERG induced expression of neurotransmitter receptors in prostate cancer cells, and a specific mechanism by which ERG may contribute to tumor development.
Expression of ERG is an early event in prostate cancer tumorigenesis, but alone is insufficient to induce cancer (3, 4). Here we investigated how ERG changed the function of prostate cancer cells by transducing LNCaP and PC3 cells with ERG, and measuring changes in gene expression and metabolic profiles. Amongst the set of genes most significantly changed, we found that expression of ERG in LNCaP and PC3 cells caused up-regulation of acetylcholine and adrenergic receptors (Fig. 1). Additionally, metabolic profiling of these cell lines revealed an increase in the concentration of excitatory neural signaling molecules, choline, glutamate and quinolinate (Fig. 3). To test the functionality of the neuro-transmitter receptors, we treated cells with the acetylcholine receptor agonist, nicotine. Signaling through these receptors induced an increase in Ca2+ release, phosphorylation of GSK3β (Ser9) and cell proliferation (Fig. 4). GSK3β phosphorylation at Ser9 is a downstream event of many signal pathways including WNT, AKT, and Ca2+ signaling . GSK3β has many effects that could contribute to tumor development. This includes its central function as a regulator of glucose metabolism and cell proliferation  and we found that metabolites within both gluconeogenesis and the citric acid cycle were two of the most enriched pathways of ERG expressing cells (Fig. 4). Additionally, metabolites involved in protein biosynthesis, purine metabolism, and pyrimidine metabolism were all increased in ERG expressing cells, suggesting a higher metabolic rate in these cells (Fig. 3). ERG has previously been reported to control phosphorylation of GSK3β (Ser9) by regulating the WNT pathway . However, our data suggests a possible novel mechanism by which ERG regulates GSK-3β; up-regulation of neuro-transmitter receptors leading to an inappropriate response to local neurotransmitters. In addition to these general roles, GSK3βhas been suggested as a contributing factor to prostate cancer cells gaining androgen independence . Together, these findings suggest that ERG may contribute to tumorigenesis through upregulating neurotransmitter receptors and increasing GSK3β phosphorylation and further downstream signaling events.
Given our findings of the increased growth response to nicotine in vitro, we investigated whether ERG had a similar effect on increasing susceptibility to nicotine in PCa patients. We found that patients who smoked and had TMRPSS2:ERG positive prostate cancer had a significantly higher number of biopsy cores positive for cancer, and these cores had a higher proportion of cancer involvement (Fig. 5). We acknowledge that the size of this dataset is very small, and the lack of data regarding the quantity and duration of the patients smoking habits, or other unknown confounding variables, make any certain claims about smoking leading to worse outcomes for prostate cancer patients who are ERG positive premature. However, this effect has been reported in many other cancers, in particular in lung cancer where nicotinic receptors regulate proliferation and inhibit apoptosis [23, 24]. More recently, similar effects have been described in bladder, breast and colon cancer [25, 26]. Based on the findings of other cancers and our in vitro data, we believe that this patient data carries more weight than an epidemiological study of this size usually would, and suggests that larger epidemiological studies should investigate the link between ERG-positive prostate cancer, smoking and prostate cancer disease outcome.
In summary, our data indicates that enhanced neurotransmitter receptor expression in ERG-expressing cells translates into elevated responsiveness to exogenously derived molecules like nicotine, or endogenous molecules, glutamate and epinephrine. Also, in silico analysis of the receptor expression data suggests that a similar responsiveness might also develop towards other endogenous molecules known to act on neurons, including dopamine, kainic acid, flupenthixol, ethanol, β-estradiol and Brain-derived neurotrophic factor. It is therefore possible that these molecules might negatively affect ERG+PCa if they are present in the tumor microenvironment. Together these data indicate that ERG has effects on cell metabolism and proliferation by increasing cell responsiveness to exogenous neurotransmitter receptor agonists such as nicotine, and possibly to endogenous neurotransmitters.
Digital rectal exam
Early Detection Research Network
High-grade prostatic intraepithelial neoplasia
National Comprehensive Cancer Network
We thank Min Yuan and Susanne Breitkopf for assistance with mass spectrometry experiments and Gregory Sanda, Daniel McManus, and Dillon Le for technical assistance. The work was partially supported by NIH grants 5P01CA120964 (J.M. Asara), 5P30CA006516 (J.M. Asara), the NIH-National Cancer Institute Early Detection Research Network grant UO1-CA113913 (M.G. Sanda), the National Cancer Institute Prostate Specialized Program of Research Excellence Career Development Award 2P50CA90381-06 (M.S. Arredouani), the Prostate Cancer Foundation Young Investigator Award (M.S. Arredouani), the Prostate Cancer Foundation A. Mazzone Challenge Award (M.G. Sanda and M.S. Arredouani), the Department of Defense Prostate Cancer Research Program New Investigator Award W81XWH-09-1-0448 (M.S. Arredouani), Department of Defense Prostate Cancer Research Program Laboratory-Clinical Transition Award W81XWH-09-1-0156 (M.S. Arredouani & M.G. Sanda, Co-PI), US Department of Defense Prostate Cancer Research Program Laboratory-Postdoctoral Training Award W81XWH-13-1-0246 (H.T. Kissick).
Novelty and impact
The TMPRSS2-ERG gene fusion occurs in about half of prostate cancer (PCa) cases and results in overexpression of the transcription factor ERG. Here we show that overexpression of ERG in prostate tumor cells increased expression of acetylcholine receptors and enhanced responsiveness to receptor agonists such as nicotine. This work offers a novel mechanism by which ERG contributes to tumorigenesis.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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