Reduction of Two Histone Marks, H3k9me3 and H3k27me3 by Epidrug Induces Neuroendocrine Differentiation in Prostate Cancer

Received: 30 November 2017 | Accepted: 6 December 2017 DOI: 10.1002/jcb.26586

RESEARCH ARTICLE

Reduction of two histone marks, H3k9me3 and H3k27me3 by epidrug induces neuroendocrine differentiation in prostate cancer

Eunsohl Lee1 | Jingcheng Wang1 | Younghun Jung1 | Frank C. Cackowski1,2 | Russell S. Taichman1

1 Department of Periodontics and Oral Medicine, University of Michigan School of Abstract Dentistry, Ann Arbor, Michigan Neuroendocrine prostate cancer (NE PCa) is an aggressive malignancy, often 2 Department of Internal Medicine, Division presenting with advanced metastasis. We previously reported that reduction of of Hematology and Oncology, University of Michigan School of Medicine, Ann Arbor, histone marks regulated by DNMT1 following epidrug (5-Azacitidine, 5-Aza) Michigan treatment controls induction of epithelial to mesenchymal (EMT) and a cancer stem cell (CSC) phenotype, which facilitates tumorigenesis in PCa cells. Here, we use the Correspondence Russell S. Taichman DMD, DMSc, epidrug 5-Aza as a model for how histone marks may regulate the reprogramming of Department of Periodontics and Oral prostate adenocarcinoma into NE phenotypic cells. First, we observed that 5-Aza Medicine, University of Michigan School of treatment of PCa cells in vitro induces a neuron-like phenotype. In addition, Dentistry, 1011 North University Avenue, Ann Arbor 48109, MI. significant increases in the expression of the NE markers N-Myc downstream Email: [email protected] regulated gene 1 (NDRG1), enolase-2 (ENO2), and synaptophysin were observed.

Funding information Critically, a high density of NE cells with synaptophysin expression was found in U.S. Department of Defense, tumors generated by 5-Aza pretreatment of PCa cells. Importantly, induction of NE Grant numbers: W81XWH-14-1-0403, differentiation of PCa cells was associated with an enhancement of NDRG1 W81XWH-15-1-0413, W81XWH-15-1- 0637; Prostate Cancer Foundation; National expression by reduction of two histone marks, H3K9me3 and H3K27me3. Further, Cancer Institute, Grant numbers: more NDRG1 expression was detected in the subset of PCa cells with reduced CA093900, CA163124 expression of H3K9me3 or H3K27me3 in the tumors generated by 5-Aza pretreated PCa cells and critically, these biological differences are also observed in small cell carcinoma in advanced stage of human primary PCa tumors. Our results suggest that reduction of histone marks regulated by the epidrug 5-Aza may control induction of a NE phenotype, which facilitates PCa progression. These studies suggest a strong rationale for developing therapeutics, which target epigenetic regulation.

KEYWORDS epidrug, histone marks, neuroendocrine, prostate cancer

1 | INTRODUCTION metastases at the time of death.2 Androgen deprivation therapy (ADT) is the primary treatment option for metastatic PCa. Prostate cancer (PCa) is the second leading cause of cancer Unfortunately, PCa cells frequently become resistant to ADT, deaths in American males.1 Most PCa metastasizes or spreads to suggesting a critical need for the identification of therapeutic bones and almost all men who die from PCa have bone targets and more effective therapies in advanced PCa disease.3,4

Neuroendocrine prostate cancer (NE PCa) is an extremely 2 | MATERIALS AND METHODS aggressive subtype of PCa that is commonly observed in relapsing individuals particularly after treatment with second- 2.1 | Cell culture generation anti-androgens (abiraterone and enzalutamide).3–9 The Human PCa cell lines, PC3 and DU145 were obtained NE PCa is characterized by neuroendocrine (NE) markers from the American Type Culture Collection (Rockville, MD). such as chromogranin A, synaptophysin, and neuron specific PCa cells were grown in RPMI 1640 (Invitrogen, Carlsbad, enolase (NSE), but no or low expression of androgen receptor CA) supplemented with 10% fetal bovine serum (FBS) and (AR) and androgen target genes such as prostate specific – 1% penicillin/streptomycin (P/S). antigen (PSA/KLK-3).3 9 NE PCa typically metastasizes to various organs such as liver, lymph node, visceral, and bone. Diagnosis of NE PCa disease is associated with a poor 2.2 | Quantitative RT-PCR prognosis with high mortality rates.3,4 Therefore, understand- Total RNA was extracted from cells using the RNeasy mini ing the mechanisms underlying the emergence of NE PCa in kit (cat. 74104, Qiagen, Valencia, CA) and converted into advanced metastasis is crucial for developing future therapies cDNA using a First-Strand Synthesis Kit (Invitrogen). for cancer treatment. Quantitative PCR (real-time PCR) was performed on an Epigenetic modifications such as DNA methylation or ABI 7700 sequence detector using TaqMan Universal PCR histone methylation, acetylation, and phosphorylation regu- Master Mix according to the directions of manufacturer late the phenotypic changes of cells by the alternation of gene (Applied Biosystems, Foster City, CA). TaqMan MGB expression without concomitant genomic changes and the probes (Applied Biosystems) were as follows: N-Myc histone modifications also play the critical roles in downstream regulated 1 (NDRG1, Hs00608387_m1), transcriptional regulation.10,11 Emerging evidence indicates Enolase-2 (ENO2, Hs00157360_m1), Synaptophysin (SYP, that the epigenetic alterations play a more fundamental Hs00300531_m1). β-Actin (Hs01060665_g1) was used as function in NE PCa initiation and development.10,12 NE PCa internal controls for the normalization of target gene disease is often associated with epigenetic alterations expression. including AR inactivation, the loss of tumor suppressors (RB1, PTEN, and TP53), TMPRSS2-ERG rearrangement, and amplification of proto-oncogenes (Aurka and 2.3 | Immunostaining N-Myc).4,10 Recent evidence has been shown that treatment with 5-Aza is associated with histone H3 lysine 9 (H3K9) Tumor sections were used on the tumors subcutaneously demethylation in some silenced genes, which results in grown with vehicle or 5-Aza pretreated PCa cells implanted transcriptional regulation.13 We previously reported that the into SCID mice in our previous studies.14 Tumor sections reduction of histone marks regulated by DNMT1 following were deparaffinized, hydrated, antigen retrieval, and then 5-Aza treatment promoted the induction of EMT and CSC blocked with Image-iT FX signal enhancer for 30 min and phenotypes, which facilitates tumorigenesis in PCa cells.14 incubated for 2 h at room temperature with primary Emerging evidence indicates that the acquisition of CSC and antibodies combined with reagents of Zenon Alexa Fluor EMT phenotypes are associated with the development of NE 488 (green) (cat. Z25002, Invitrogen) or 555 (red) (cat. cell differentiation in PCa, leading to more aggressive Z25305, Invitrogen) labeling kit. Synaptophysin (cat. disease.3,4,15,16 Here, we use 5-Aza to further explore whether ab23754, Abcam), Pan-Cytokeratin (cat. ab86734, Abcam), reduction of histone marks may regulate the reprogramming N-Myc downstream regulated 1 (NDRG1) (cat. ab196621, of PCa cells into NE PCa cells. Abcam), H3K9me3 (cat. ab6001, Abcam), and H3K27me3 We observed that PCa cells alter their morphological (cat. ab6147, Abcam) antibodies were used as primary features to a neuron-like phenotype after 5-Aza treatment in antibody. After washing with PBS, the slides were mounted vitro. We also found that a significant increase in the expression with ProLong Gold antifade reagent with DAPI (cat. of NE markers, N-Myc downstream regulated gene 1 (NDRG1), P36931, Invitrogen). H&E stain, Synaptophysin (cat. ab enolase-2 (ENO2), and synaptophysin following 5-Aza treat- 2375, Abcam), pan-Cytokeratin (cat. ab86734, Abcam) ment. Critically, in an animal model it was observed that a high antibodies were used for immunohistochemical (IHC) density of NE cells with synaptophysin expression were found in staining in s.c. tumors. Images were taken with Olympus tumors generated by pretreatment with 5-Aza. We further show 51A microscope. Co-staining of H3K9me3 or H3K27me3 that enhancement of NDRG1expression by reduction of histone with NDRG1 was applied to s.c. tumors. Images were taken marks is associated in induction of NE differentiation of PCa with Olympus FV-500 confocal microscope. Human cells. Our findings suggest that interference of these pathways prostate tissue microarrays (TMAs) were purchased from may be possible ways for the targeting NE development and US Biomax, Inc. (Rockville, MD). Tumors were graded progression. LEE ET AL. | 3699 using stage progressing system. Co-staining of H3K9me3 or tests were conducted on data sets of 2 comparisons, and H3K27me3 with NDRG1 was applied to TMAs. significance of data with multiple comparisons was evaluated using an ANOVA. Values of P < 0.05 were 2.4 | Western blot considered significant.

PCa cells were cultured in RPMI 1640 with 10% FBS and 1% 3 | RESULTS P/S. The cells were treated with vehicle or 5-Aza (5 μM) for 10 days at 37°C. Whole cell lysates were prepared from cells, 3.1 | Epidrug induces a neuroendocrine-like separated on 4-20% Tris-Glycine gel and transferred to PVDF phenotype of PCa cells in vitro membranes. The membranes were incubated with 5% milk for 1 h and incubated with primary antibodies overnight at 4°C. To explore the role that the epigenetic alterations play in the Primary antibodies used were as follows: N-Myc downstream development of neuroendocrine (NE) differentiation, we regulated 1 (NDRG1, 1:1000 dilution, cat. 9485, Cell examined the morphological changes in PCa cells follow- Signaling), Synaptophysin (1:1000 dilution, cat. 4329, Cell ing 10 days of 5-Aza treatment. We found that 5-Aza Signaling,), Enolase-2 (ENO2, 1:1000 dilution, cat. 9536, treatment of PCa cells (PC3, DU145) results in the Cell Signaling,), H3K9me3 (1:1000 dilution, cat. 13969, Cell development of a neuroendocrine-like phenotype (Figure 1). Signaling), and H3K27me3 (1:1000 dilution, cat. 9733, Cell Specifically, 5-Aza treated cells were developed a neuro- Signaling). Blots were incubated with peroxidase-coupled nal-like appearance, which is characterized by bipolar or anti-rabbit IgG secondary antibody (cat. 7074, 1:2000 multipolar cells with small cell bodies, long-projecting dilution, Cell Signaling) for 1 h, and protein expression axonal processes. In contrast, vehicle treated cells were was detected with SuperSignal West Dura Chemiluminescent maintained their epithelial morphology and tended to grow Substrate (cat. Prod 34075, Thermo Scientific, Rockford, IL). in clusters (Figure 1). The data suggest that 5-Aza treatment Membranes were reprobed with monoclonal anti-β-Actin alone may be a sufficient stimulus to induce epigenetic antibody (1:1000 dilution, cat. 4970, Cell Signaling) to alteration in PCa cell lines to induce a neuroendocrine-like control for equal loading. phenotype.

2.5 | In vitro chromatin immunoprecipitation 3.2 | Epidrug increases expression of (CHIP) neuroendocrine phenotypic marker in PCa cells in vitro CHIP assays were performed on the vehicle or 5-Aza (5 μM) treated PCa cells (PC3 or DU145) (4 × 106)by To determine if the epigenetic alteration by the epidrug 5-Aza following the directions of the manufacturer (cat. 334471, regulates NE differentiation, we first examined the expression EpiTech CHIP OneDay Kit, Qiagen). PCa cells were treated of NE phenotypes in vehicle- or 5-Aza treated PCa cells by with 1% formaldehyde to crosslink histones to DNA. The real-time PCR and Western blots. Expression of mRNA for crosslinking was stopped by treating the samples with stop the NE associated transcription factor, N-Myc downstream buffer for 5 min. The chromatin was extracted and regulated gene 1 (NDRG1) was increased in 5-Aza treated fragmented by sonication, and the lysate was used to PCa cells versus vehicle treated PCa cells (Figure 2A). immunoprecipitation using Protein A + G beads and the Likewise, NE markers enolase-2 (ENO2) and synaptophysin following antibodies; H3K9me3 (cat. GAH-6204, Qiagen), were significantly increased by 5-Aza treatment, indicating H3K27me3 (cat. GAH-9205, Qiagen), or Rabbit control the induction of a NE phenotype (Figures 2B and 2C). At the IgG (cat. GAH-9205, Qiagen). Immunocomplexes were protein level, a significant increase in NDRG1, ENO2, and pulled down, washed, and DNA was isolated to run SYBR synaptophysin expression was observed in response to 5-Aza qPCR with NDRG1 (assay tile: −0.4Kb; assay position: treatment compared to vehicle treatment (Figure 2D). These −3262) (cat. GPH1026403(−)04A, Qiagen) specific primer data suggest that epigenetic alteration by 5-Aza is associated on promoter site. with a NE phenotype in PCa cells.

2.6 | Statistical analyses 3.3 | Epidrug induces an neuroendocrine phenotype in PCa tumor cells in vivo Results are presented as mean ± standard deviation (s.d.). Distribution of the data was determined by skewness. Our previous studies demonstrate that significantly greater Values within a 1.95 (5%) confidence interval were tumor sizes were observed in SCID mice implanted with PCa accepted as a normal distribution and evaluated further cells pretreated with 5-Aza in vitro compared to PCa cells using parametric tests for significance. Unpaired Student's t- pretreated with vehicle.14 Here, we found that a significantly 3700 | LEE ET AL.

FIGURE 1 Induction of a neuroendocrine-like phenotype by epidrug (5-Aza) in PCa cells in vitro. PC3 or DU145 cells (1 × 105) were treated for 10 days with vehicle or 5-Aza (5 μM) and subsequently imaged by bright field microscopy. Bar = 100 μm higher density of NE PCa cells with extensive vascularization vehicle (Figures 3A and 3B). Further, to confirm that the (Figures 3A and 3B, H&E stain) and synaptophysin tumor cells were of human orign, Pan-Cytokeratin expression expression in tumors generated from PCa cells pretreated was examined (Figures 3A and 3B). These data suggest that with 5-Aza in vitro compared to PCa cells pretreated with the epigenetic alteration by the epidrug 5-Aza may regulate

FIGURE 2 Increase of expression of neuroendocrine phenotypic marker by epidrug (5-Aza) in PCa cells. PCa cells (PC3 or DU145) (2 × 105) were seeded in 10 cm culture dishes. The cells were treated by vehicle or 5-Aza (5 μM) for 10 days. A-C, mRNA levels of N-Myc downstream regulated 1 (NDRG1), Enolase-2 (ENO2), and Synaptophysin expression in vehicle or 5-Aza (5 μM) treated PCa cells (PC3 or DU145) as quantified by real-time PCR. The data in (A-C) are representative of mean ± s.d (n = 6). P values were calculated by Student's t-test. D, Protein levels of NDRG1, ENO2, and Synaptophysin following vehicle or 5-Aza (5 μM) treatments in PCa cells (PC3 or DU145) as quantified by Western blots LEE ET AL. | 3701

FIGURE 3 Induction of neuroendocrine phenotypic cells by epidrug (5-Aza) in PCa tumors in vivo. Examination of NE markers in tumors grown in SCID mice implanted s.c. with PCa cells pretreated with vehicle or 5-Aza in vitro.14 High density of NE PCa cells in (A) PC3 tumors and (B) DU145 tumors were shown by H&E staining (NE PCa: red) with an indication of black arrow. Bar = 200 μm. Synaptophysin or Pan-Cytokeratin expressing NE PCa cells as detected by IHC staining. Bar = 50 μm the induction of a NE phenotype, leading to the emergence of treated with 5-Aza or vehicle by Western blots. We found that malignant outgrowth. the levels of both suppressive histone marks, H3K9me3 and H3K27me3, were significantly reduced in PCa cells treated with 3.4 | Epidrug induces the reduction of histone 5-Aza compared to PCa cells treated with vehicle (Figure 4A). marks on the NDRG1 promoter in PCa cells We further examined whether reduction of suppressive histone marks, H3K9me3 and H3K27me3 on NDRG1 5-Aza regulates global reductions of di- and tri-methylation of promoter induce NDRG1 expression in the 5-Aza or vehicle histone H3 lysine 9 (H3K9me2 and H3K9me3) in the many treated PCa cells by CHIP qPCR assays (Figures 4B and 4C). human cancer cells.13 We therefore examined whether 5-Aza In both cases, we observed that the levels of suppressive induces reduction of tri-methylation of histone H3 lysine 9 histone marks were significantly suppressed in the promotor (H3K9me3) and histone H3 lysine 27 (H3K27me3) in PCa cells of NDRG1 site (−0.4Kb [−3262]) in 5-Aza treated PCa cells, 3702 | LEE ET AL.

FIGURE 4 Reduction of histone marks on the NDRG1 promoter by epidrug (5-Aza) in PCa cells. A, Examination of global changes of histone silent marks, H3K9me3, and H3K27me3 in PCa cells (PC3, DU145) following in vehicle or 5-Aza treated PCa cells by Western blots. B, Experimental design of CHIP qPCR assays in vehicle or 5-Aza treated PCa cells. C, A scheme shows the primer site at the promoter region from transcription start site of the human NDRG1 genomic locus. D, % respective input of H3K9me3 and H3K27me3 levels at the NDRG1 genomic locus as determined by CHIP qPCR assays in vehicle or 5-Aza treated PCa cells (PC3, DU145). The data in (D) are representative of mean ± s.d. (n =3). P values are calculated by Student's t-test. E, A summary diagram shows the activation of chromatin by histone demethylation of H3K9me3 and H3K27me3 following 5-Aza treatment, resulting in the increase of NDRG1 transcription which promote the activation of chromatin, resulting in the H3K27me3 expression, while more NDRG1 expression was increase of transcription of NDRG1 (Figures 4D and 4E). The detected in the subset of H3K9me3 or H3K27me3 reduced data suggest that reduction of suppressive histone marks on cell populations in 5-Aza pretreated PCa tumors (Figures the NDRG1 promoter is important in the regulation of 5A and 5B). NDRG1 transcription in the induction of a NE phenotype. To examine whether our cell line data is indicative of what happens in human tumors, staining for H3K9me3 or 3.5 | Reduction of two histone marks, H3K27me3 with NDRG1 was performed using tissue H3k9me3 and H3k27me3, associate with microarrays (TMA). TMAs from PCa patients, it was NDRG1 in PCa tumors and human primary demonstrated that significant levels of H3K9me3 or PCa tumors H3K27me3 expression in high grade tumor cells compare to normal prostate epithelial tissues or low grade tumor cells. To further determine whether NE phenotypic changes by Critically, high levels of H3K9me3 or H3K27me3 expression reduction of histone marks in vitro reflects the activity in vivo, are detected in advanced cancer cells (Figures 5C and 5D). tumors grown s.c. were stained for H3K9me3 or H3K27me3 We further found that high levels of H3K9me3 or H3K27me3 with NDRG1. Tumors generated from PCa cells with expression with low NDRG1 expression in the luminal type of pretreated vehicle showed high levels of H3K9me3 or adenocarcinoma cells, while suppressed H3K9me3 or LEE ET AL. | 3703

FIGURE 5 Association of H3K9me3 and H3K27me3 reduction with NDRG1 in s.c. PCa tumors and human primary PCa tumors. A, NDRG1 (green) expression (white arrow) in H3K9me3 (red) or H3K27me3 (red) reduced cells in vehicle or 5-Aza pretreated s.c. PCa tumors as detected by immunofluorescence co-staining. Blue, DAPI nuclear stain. Bar = 20 μm. B, Quantification of (A). Data represent as mean ± s.d. (n = 8/group) (Student's t-test). C, NDRG1 (green) expression (white arrow) in H3K9me3 (red) or H3K27me3 (red) expressing cells in human primary PCa tumors from the tissue microarray samples (TMA) from PCa patients as detected by immunofluorescence co-staining. Blue, DAPI nuclear stain. Bar = 20 μm. TMAs are normal prostate tissue (n = 8), Gleason 6 prostate cancer tissue (n = 9), and Gleason 9 prostate cancer tissue (n = 5). D, Quantification of (C). Data represent as mean ± s.d. (Student's t-test)

H3K27me3 expression with an enhanced NDRG1 expression enhanced NDRG1 expression suggests that epigenetic in small cell carcinoma cells in advanced stage of human regulation of gene expression plays a critical role in the primary PCa tumors, which correlated with our findings in development of aggressive disease. s.c. animal models in this study. The data suggest that NE PCa is correlated with prostate tumor progression reduction of histone marks is associated with more NDRG1 representing the poor prognosis with lethal events.3–9 expression in the NE phenotypic cells, which plays a critical Although androgen deprivation therapy (ADT) provides role in PCa tumor progression.

4 | DISCUSSION

Neuroendocrine prostate cancer (NE PCa) is an aggressive malignancy, often observed in relapsing individuals particularly after second generation of androgen- deprivation therapy in advanced metastasis. Here, we show that epigenetic alteration by the epidrug 5-Aza may associate with the reprogramming of prostate adenocarcinoma cells into NE phenotypic PCa cells. Our studies FIGURE 6 Experimental Model. Reduction of histone marks by demonstrate that induction of NE differentiation of PCa the epidrug 5-Aza regulates induction of NE phenotype, which cells through reduction of histone marks leading to facilitates the tumor progression in PCa cells 3704 | LEE ET AL. most patients early clinical responses, this approach Growing evidence shows that deregulation of cellular remains the limitation for patients with the advanced signaling by genetic or epigenetic alterations are associ- metastatic PCa, subsequently developing resistance to ated with neuroendocrine differentiation.10 Like what we these treatments.3–9 observed in PCa cells, there are growing evidences that the There are at least two possible mechanisms underlying demethylation agent, 5-Aza induces N-myc downstream- the development of NE PCa disease. One possibility is the regulated gene 1 (NDRG1) expression in pancreatic emergence of metastatic tumor clones or cells with tumor cancer, breast cancers, and colon cancer, which associated initiating potential or CSCs that gave rise to at least three in angiogenesis, metastases, and mechanisms leading to distinct components (luminal, basal, and NE), leading anti-cancer drug resistance.22–26 A recent report also extraordinarily aggressive during tumor progression.4 NE shows that HIF-1α upregulates NDRG1expression through cells represent a less differentiated phenotype sharing binding to NDRG1 promoter, leading to proliferation of several CSC characteristics, which are distinct from the lung cancer A549 cells22 and NDRG1 expression is highly differentiated luminal type of adenocarcinoma associated with metastasis and neuroendocrine differenti- cells.3 For example, NE cells express high levels of the ation in PCa cells through loss of RB in a hypoxia- stem cell surface marker PROM1/CD133.3 In fact, a recent dependent manner manner.27 Together, these studies study shows the association NE PCa cells with CSC and/or support our findings that reduction of histone marks on EMT cells that were confirmed by the injection of CSCs the NDRG1 promoter by 5-Aza regulates the NDRG1 into nude mice, which generates highly vascularized transcription, which contributes to induction of a NE tumors showing high density of NE PCa cells and phenotype in PCa cells. expressing low levels of E-cadherin and β-catenin and In summary, these studies evidence that reduction of high levels of vimentin.16 Moreover a further study histone marks by the epidrug 5-Aza regulates induction of demonstrates a direct link between EMT and NE whereby NE phenotype, which facilitates the tumor progression in the transcription factor snail, which normally induces PCa cells (Figure 6). Thus, these studies have important EMT, also may induce a NE phenotype.15 implications for pre-clinical studies of epigenetic targeted A second pathway for the emergence of NE disease is therapies. Clinically, 5-Aza-CdR continues to be an through transdifferentiation of preexisting adenocarci- effective agent for growth inhibition and induction of noma cells, which acquire several molecular alterations.4 apoptosis during the chemotherapy in hematologic malig- In these biologic processes, cell autonomous signaling nancies and in several solid tumors including PCa pathways facilitate acquisition of a NE state, which in tumors.28,29 In previous studies, we demonstrated that conjunction with microenvironmental clues, promote reduction of DNMT1 by 5-Aza is associated with the tumor outgrowth, survival, and therapeutic-resistant prop- emergence of CSC and EMT phenotypes, suggesting that erties. As examples, IL-6, epinephrine, and forskolin several potential side-effects are possible with the thera- induce NE differentiation in PCa cells through activation peutic use of 5-Aza in prostate cancer. Our data suggest that of intracellular cAMP and protein kinase A (PKA).17–19 careful monitoring and evaluation of dose and treatment Further, CXCL12 and its receptors (CXCR4, CXCR7) are schedules are warranted for use of 5-Aza-CdR in anticancer actively expressed in NE cells and signals on mTOR.20 therapies. Neuropeptides secreted by NE cells activate Src, ERK, and PI3 K/Akt that can act in a paracrine manner to stimulate ACKNOWLEDGMENTS growth, survival, motility, and metastatic potential of androgen-independent epithelial PCa cells.4 Together This work was supported by the National Cancer Institute these studies suggest that multiple signaling pathways (NIH/NCI PO1-CA093900, the NIH/NCI Tumor Microenvi- are involved in NE differentiation, ultimately contributing ronment Network U54-CA163124 and supplement), the to PCa progression. It has also been considered that, the Department of Defense (W81XWH-14-1-0403, W81XWH- NE transdifferentiation process may be an adaptation 15-1-0637, W81XWH-15-1-0413), and the Prostate Cancer mechanism, PCa cells allow to adapt a wide spectrum of Foundation. R.T. receives support as the Major McKinley therapeutic agents.4,10 Likewise, accumulation of new Ash Collegiate Professor. genetic alterations such as loss of tumor suppressors (RB1, PTEN, and TP53), TMPRSS2-ERG rearrangements, AUTHOR’SCONTRIBUTIONS amplifications of proto-oncogenes (Aurka and N-Myc) under evolutionary pressures or through the natural EL and RST designed experiments. EL, JW, YJ, and FCC. selection, may facilitate the development of the NE small performed experiments and analyzed the data. Dr YL cell carcinoma phenotype, which promotes extremely discussed the results and gave valuable critique on the paper. tumor outgrowth with rapid proliferative activity.4,10,21 EL and RST wrote the manuscript. LEE ET AL. | 3705

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